Pharmaceutical Compositions with Attenuated Release of Phenolic Opioids

ABSTRACT

Pharmaceutical compositions and their methods of use are provided, where the pharmaceutical compositions comprise a phenolic opioid prodrug that provides enzymatically-controlled release of a phenolic opioid, and an enzyme inhibitor that interacts with the enzyme(s) that mediates the enzymatically-controlled release of the phenolic opioid from the prodrug so as to attenuate enzymatic cleavage of the prodrug.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority benefit under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/106,400, filed on Oct. 17, 2008.

INTRODUCTION

Phenolic opioids are susceptible to abuse. Access to these drugstherefore needs to be controlled. The control of access to the drugs isexpensive to administer and can result in denial of treatment forpatients that are not able to present themselves for dosing. Forexample, patients suffering from acute pain may be denied treatment withan opioid unless they have been admitted to a hospital.

International patent application, publication number WO 2007/140272describes certain prodrugs that afford controlled release of phenolicopioids. The prodrugs are resistant to abuse, being stable in thepresence of household chemicals such as vinegar or baking soda, andrequire enzyme activation in the gut to initiate release of the phenolicopioid. The prodrugs are believed to release the phenolic opioid throughan enzyme-activated cyclisation release mechanism. Thus, enzyme-inducedcleavage of an amide bond is believed to afford a nucleophilic nitrogenatom, which then undergoes a cyclisation-release reaction.

The prodrugs described in WO 2007/140272 resist releasing phenolicopioid when subjected to conditions commonly used by those who wish toabuse the drug, but release phenolic opioid when administered orally.This provides substantial protection against abuse. However, there aresituations in which oral consumption of such a prodrug could potentiallyresult in overexposure to the phenolic opioid, whether by abuse oraccidental over-consumption.

SUMMARY

The present disclosure provides pharmaceutical compositions, and theirmethods of use, where the pharmaceutical compositions comprise aphenolic opioid prodrug that provides enzymatically-controlled releaseof a phenolic opioid, and an enzyme inhibitor that interacts with theenzyme(s) that mediates the enzymatically-controlled release of thephenolic opioid from the prodrug so as to attenuate enzymatic cleavageof the prodrug.

According to one aspect, therefore, the embodiments of the inventioninclude pharmaceutical compositions, which comprise a trypsin inhibitorand a compound of general formula (I):

X—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵)  (I)

or a pharmaceutically acceptable salt thereof, in which:

X represents a residue of a phenolic opioid, wherein the hydrogen atomof the phenolic hydroxyl group is replaced by a covalent bond to—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵);

R¹ represents a (1-4C)alkyl group;

R² and R³ each independently represents a hydrogen atom or a (1-4C)alkylgroup;

n represents 2 or 3;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group, or a residue of an aminoacid, a dipeptide, or an N-acyl derivative of an amino acid ordipeptide.

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general formula (II):

X—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵)  (II)

or a pharmaceutically acceptable salt thereof, in which:

X represents a residue of a phenolic opioid, wherein the hydrogen atomof the phenolic hydroxyl group is replaced by a covalent bond to—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵);

R¹ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R² and R³ together with the carbon to which they are attached form acycloalkyl and substituted cycloalkyl group, or two R² or R³ groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n represents an integer from 2 to 4;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group (including N-substitutedacyl), a residue of an amino acid, a dipeptide, an N-acyl derivative(including N-substituted acyl derivative) of an amino acid or dipeptide.

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general formula (III):

X—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵)  (III)

or pharmaceutically acceptable salt thereof, in which:

X represents a residue of a phenolic opioid, wherein the hydrogen atomof the phenolic hydroxyl group is replaced by a covalent bond to—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵);

R¹ represents a (1-4C)alkyl group;

-   -   R² and R³ each independently represents a hydrogen atom or a        (1-4C)alkyl group;    -   n represents 2 or 3;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group (including N-substitutedacyl), a residue of an amino acid, a dipeptide, an N-acyl derivative(including N-substituted acyl derivative) of an amino acid or dipeptide.

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general formula (IV):

or pharmaceutically acceptable salt thereof, in which:

R^(a) is hydrogen or hydroxyl;

R^(b) is oxo (═O) or hydroxyl;

the dashed line is a double bond or single bond;

R¹ represents a (1-4C)alkyl group;

R² and R³ each independently represents a hydrogen atom or a (1-4C)alkylgroup;

n represents 2 or 3;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group, or a residue of an aminoacid, a dipeptide, or an N-acyl derivative of an amino acid ordipeptide.

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general formula (V):

or pharmaceutically acceptable salt thereof, in which:

R^(a) is hydrogen or hydroxyl;

R^(b) is oxo (═O) or hydroxyl;

the dashed line is a double bond or single bond;

R¹ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R² and R³ together with the carbon to which they are attached form acycloalkyl and substituted cycloalkyl group, or two R² or R³ groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n represents an integer from 2 to 4;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group (including N-substitutedacyl), a residue of an amino acid, a dipeptide, an N-acyl derivative(including N-substituted acyl derivative) of an amino acid or dipeptide.

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general formula (VI):

or pharmaceutically acceptable salt thereof, in which:

R^(a) is hydrogen or hydroxyl;

R^(b) is oxo (═O) or hydroxyl;

the dashed line is a double bond or single bond;

R¹ represents a (1-4C)alkyl group;

R² and R³ each independently represents a hydrogen atom or a (1-4C)alkylgroup;

n represents 2 or 3;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group (including N-substitutedacyl), a residue of an amino acid, a dipeptide, an N-acyl derivative(including N-substituted acyl derivative) of an amino acid or dipeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph that compares mean blood concentrations over time ofhydromorphone (HM) following PO administration to rats of Compound 1alone and Compound 1 with various amounts of trypsin inhibitor fromGlycine max (soybean) (SBTI).

FIG. 2 is a graph that compares mean plasma concentrations over time ofhydromorphone (HM) following PO administration to rats of Compound 1alone, Compound 1 with ovalbumin (OVA), and Compound 1 with ovalbuminand SBTI.

FIG. 3 is a graph that compares individual blood concentrations overtime of hydromorphone (HM) following PO administration to rats ofCompound 1 alone and Compound 1 with Bowman-Birk trypsin-chymotrypsininhibitor (BBSI).

FIG. 4 is a graph that compares mean plasma concentrations over time ofhydromorphone (HM) release following PO administration of Compound 2alone and Compound 2 with SBTI to rats.

FIG. 5 is a graph that compares mean plasma concentrations over time ofhydromorphone (HM) release following PO administration of Compound 3alone and Compound 3 with SBTI to rats.

FIG. 6 is a graph that compares mean plasma concentrations over time ofhydromorphone (HM) release following PO administration of Compound 4alone and Compound 4 with SBTI to rats.

FIGS. 7A and 7B are graphs that indicate the results of exposure of acertain combination of Compound 4 and trypsin, in the absence of anytrypsin inhibitor or in the presence of SBTI, Compound 107, Compound108, or Compound 109. FIG. 7A depicts the disappearance of Compound 4,and FIG. 7B depicts the appearance of hydromorphone, over time underthese conditions.

FIG. 8 is a graph that compares mean plasma concentrations over time ofhydromorphone (HM) release following PO administration of Compound 3alone and Compound 3 with. Compound 101 to rats.

FIG. 9 is a graph that compares mean plasma concentrations over time ofhydromorphone (HM) release following PO administration of Compound 4alone and Compound 4 with Compound 101 to rats.

DEFINITIONS

The following terms have the following meaning unless otherwiseindicated. Any undefined terms have their art recognized meanings.

As used herein, the term “alkyl” by itself or as part of anothersubstituent refers to a saturated branched or straight-chain monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Typical alkyl groups include, butare not limited to, methyl; ethyl, propyls such as propan-1-yl orpropan-2-yl; and butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl or 2-methyl-propan-2-yl. In some embodiments, analkyl group comprises from 1 to 20 carbon atoms. In other embodiments,an alkyl group comprises from 1 to 10 carbon atoms. In still otherembodiments, an alkyl group comprises from 1 to 6 carbon atoms, such asfrom 1 to 4 carbon atoms.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The groupmay be in either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to, ethenyl;propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc.; and the like.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Typicalalkynyl groups include, but are not limited to, ethynyl; propynyls suchas prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Acyl” by itself or as part of another substituent refers to a radical—C(O)R³⁰, where R³° is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as definedherein. Representative examples include, but are not limited to formyl,acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl,benzylcarbonyl, piperonyl, and the like. Substituted acyl refers tosubstituted versions of acyl and include, for example, but not limitedto, succinyl and malonyl.

The term “aminoacyl” and “amide” refers to the group —C(O)NR²¹R²²,wherein R²¹ and R²² independently are selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic and where R²¹ and R²² are optionally joined together withthe nitrogen bound thereto to form a heterocyclic or substitutedheterocyclic group, and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Alkoxy” by itself or as part of another substituent refers to a radical—OR³¹ where R³¹ represents an alkyl or cycloalkyl group as definedherein. Representative examples include, but are not limited to,methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.

“Alkoxycarbonyl” by itself or as part of another substituent refers to aradical —C(O)OR³¹ where R³¹ represents an alkyl or cycloalkyl group asdefined herein. Representative examples include, but are not limited to,methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,cyclohexyloxycarbonyl and the like.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent aromatic ringsystem. Typical aryl groups include, but are not limited to, groupsderived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene and the like. In some embodiments, an arylgroup comprises from 6 to 20 carbon atoms. In other embodiments, an arylgroup comprises from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenyleth-1-yl, naphthylmethyl, 2-naphthyleth-1-yl,naphthobenzyl, 2-naphthophenyleth-1-yl and the like. In someembodiments, an arylalkyl group is (C₇-C₃₀) arylalkyl, e.g., the alkylmoiety of the arylalkyl group is (C₁-C₁₀) and the aryl moiety is(C₆-C₂₀). In other embodiments, an arylalkyl group is (C₇-C₂₀)arylalkyl, e.g., the alkyl moiety of the arylalkyl group is (C₁-C₈) andthe aryl moiety is (C₆-C₁₂).

Compounds may be identified either by their chemical structure and/orchemical name. The compounds described herein may contain one or morechiral centers and/or double bonds and therefore, may exist asstereoisomers, such as double-bond isomers (i.e., geometric isomers),enantiomers or diastereomers. Accordingly, all possible enantiomers andstereoisomers of the compounds including the stereoisomerically pureform (e.g., geometrically pure, enantiomerically pure ordiastereomerically pure) and enantiomeric and stereoisomeric mixturesare included in the description of the compounds herein. Enantiomericand stereoisomeric mixtures can be resolved into their componentenantiomers or stereoisomers using separation techniques or chiralsynthesis techniques well known to the skilled artisan. The compoundsmay also exist in several tautomeric forms including the enol form, theketo form and mixtures thereof. Accordingly, the chemical structuresdepicted herein encompass all possible tautomeric forms of theillustrated compounds. The compounds described also include isotopicallylabeled compounds where one or more atoms have an atomic mass differentfrom the atomic mass conventionally found in nature. Examples ofisotopes that may be incorporated into the compounds disclosed hereininclude, but are not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O,etc. Compounds may exist in unsolvated forms as well as solvated forms,including hydrated forms. Certain compounds may exist in multiplecrystalline or amorphous forms. In general, all physical forms areequivalent for the uses contemplated herein and are intended to bewithin the scope of the present disclosure.

“Cycloalkyl” by itself or as part of another substituent refers to asaturated cyclic alkyl radical. Typical cycloalkyl groups include, butare not limited to, groups derived from cyclopropane, cyclobutane,cyclopentane, cyclohexane and the like. In some embodiments, thecycloalkyl group is (C₃-C₁₀) cycloalkyl. In other embodiments, thecycloalkyl group is (C₃-C₇) cycloalkyl.

“Cycloheteroalkyl” by itself or as part of another substituent, refersto a saturated cyclic alkyl radical in which one or more carbon atoms(and any associated hydrogen atoms) are independently replaced with thesame or different heteroatom. Typical heteroatoms to replace the carbonatom(s) include, but are not limited to, N, P, O, S, Si, etc. Typicalcycloheteroalkyl groups include, but are not limited to, groups derivedfrom epoxides, azirines, thiiranes, imidazolidine, morpholine,piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine and thelike.

“Heteroalkyl, Heteroalkenyl and Heteroalkynyl” by themselves or as partof another substituent refer to alkyl, alkenyl and alkynyl groups,respectively, in which one or more of the carbon atoms (and anyassociated hydrogen atoms) are independently replaced with the same ordifferent heteroatomic groups. Typical heteroatomic groups which can beincluded in these groups include, but are not limited to, —O—, —S—,—O—O—, —S—S—, —O—S—, —NR³⁷R³⁸—, ═N—N═, —N═N—, —N═N—NR³⁹R⁴⁰, —PR⁴¹—,—P(O)₂—, —POR⁴²—, —O—P(O)₂—, —SO—, —SO₂—, —SnR⁴³R⁴⁴— and the like, whereR³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹, R⁴², R⁴³ and R⁴⁴ are independently hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl,substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl or substitutedheteroarylalkyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a parent heteroaromatic ring system. Typicalheteroaryl groups include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike. In some embodiments, the heteroaryl group is from 5-20 memberedheteroaryl. In other embodiments, the heteroaryl group is from 5-10membered heteroaryl. In still other embodiments, heteroaryl groups arethose derived from thiophene, pyrrole, benzothiophene, benzofuran,indole, pyridine, quinoline, imidazole, oxazole and pyrazine.

“Heteroarylalkyl” by itself or as part of another substituent, refers toan acyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. In some embodiments, the heteroarylalkyl group is a6-30 membered heteroarylalkyl, e.g., the alkyl moiety of theheteroarylalkyl is 1-10 membered and the heteroaryl moiety is a5-20-membered heteroaryl. In other embodiments, the heteroarylalkylgroup is 6-20 membered heteroarylalkyl, e.g., the alkyl moiety of theheteroarylalkyl is 1-8 membered and the heteroaryl moiety is a5-12-membered heteroaryl.

“Opioid” refers to a chemical substance that exerts its pharmacologicalaction by interaction at opioid receptors. “Phenolic opioid” refers to asubset of the opioids that contains a phenol group. Examples of phenolicopioids are provided below.

“Parent Aromatic Ring System” by itself or as part of anothersubstituent, refers to an unsaturated cyclic or polycyclic ring systemhaving a conjugated π electron system. Specifically included within thedefinition of “parent aromatic ring system” are fused ring systems inwhich one or more of the rings are aromatic and one or more of the ringsare saturated or unsaturated, such as, for example, fluorene, indane,indene, phenalene, etc. Typical parent aromatic ring systems include,but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene and the like.

“Parent Heteroaromatic Ring System” by itself or as part of anothersubstituent, refers to a parent aromatic ring system in which one ormore carbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom. Typical heteroatoms toreplace the carbon atoms include, but are not limited to, N, P, O, S,Si, etc. Specifically included within the definition of “parentheteroaromatic ring systems” are fused ring systems in which one or moreof the rings are aromatic and one or more of the rings are saturated orunsaturated, such as, for example, arsindole, benzodioxan, benzofuran,chromane, chromene, indole, indoline, xanthene, etc. Typical parentheteroaromatic ring systems include, but are not limited to, arsindole,carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene and thelike.

“Pharmaceutical composition” refers to at least one compound and apharmaceutically acceptable vehicle, with which the compound isadministered to a patient.

“Pharmaceutically acceptable salt” refers to a salt of a compound, whichpossesses the desired pharmacological activity of the parent compound.Such salts include: (1) acid addition salts, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound isreplaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike.

The term “solvate” as used herein refers to a complex or aggregateformed by one or more molecules of a solute, i.e. a compound of theembodiments or a pharmaceutically-acceptable salt thereof, and one ormore molecules of a solvent. Such solvates are typically crystallinesolids having a substantially fixed molar ratio of solute and solvent.Representative solvents include by way of example, water, methanol,ethanol, isopropanol, acetic acid, and the like. When the solvent iswater, the solvate formed is a hydrate.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with, or in which a compound is administered.

“Patient” includes humans, and also other mammals, such as livestock,zoo animals and companion animals, such as a cat, dog or horse.

“Preventing” or “prevention” or “prophylaxis” refers to a reduction inrisk of occurrence of a condition, such as pain.

“Prodrug” refers to a derivative of an active agent that requires atransformation within the body to release the active agent. Prodrugs arefrequently, although not necessarily, pharmacologically inactive untilconverted to the active agent.

“Promoiety” refers to a form of protecting group that when used to maska functional group within an active agent converts the active agent intoa prodrug. Typically, the promoiety will be attached to the drug viabond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.

“Protecting group” refers to a grouping of atoms that when attached to areactive functional group in a molecule masks, reduces or preventsreactivity of the functional group. Examples of protecting groups can befound in Green et al., “Protective Groups in Organic Chemistry,” (Wiley,2^(nd) ed. 1991) and Harrison et al., “Compendium of Synthetic OrganicMethods,” Vols. 1-8 (John Wiley and Sons, 1971-1996). Representativeamino protecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“SES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxy protecting groups include,but are not limited to, those where the hydroxy group is either acylatedor alkylated such as benzyl, and trityl ethers as well as alkyl ethers,tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Typical substituents include, but are not limited to, alkylenedioxy(such as methylenedioxy), —M, —R⁶⁰, —O⁻, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, —NR⁶⁰, CF₃, CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, C(O)O⁻,—C(S)OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹, —NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and—C(NR⁶²)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹, R⁶²and R⁶³ are independently hydrogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring.

“Treating” or “treatment” of any condition, such as pain, refers, incertain embodiments, to ameliorating the condition (i.e., arresting orreducing the development of the condition). In certain embodiments“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may not be discernible by the patient. In certainembodiments, “treating” or “treatment” refers to inhibiting thecondition, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In certain embodiments, “treating” or “treatment”refers to delaying the onset of the condition.

“Therapeutically effective amount” means the amount of a compound that,when administered to a patient for preventing or treating a conditionsuch as pain, is sufficient to effect such treatment. The“therapeutically effective amount” will vary depending on the compound,the condition and its severity and the age, weight, etc., of thepatient.

DETAILED DESCRIPTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It should be understood that as used herein, the term “a” entity or “an”entity refers to one or more of that entity. For example, a compoundrefers to one or more compounds. As such, the terms “a”, “an”, “one ormore” and “at least one” can be used interchangeably. Similarly theterms “comprising”, “including” and “having” can be usedinterchangeably.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

The nomenclature used herein to name the subject compounds isillustrated in the Examples herein. When possible, this nomenclature hasgenerally been derived using the commercially-available AutoNom software(MDL, San Leandro, Calif.).

It has now been found that attenuated release of a phenolic opioid canbe achieved by administering a trypsin inhibitor derived from soybean incombination with a particular prodrug described in WO 2007/140272.

Representative Embodiments

The present disclosure provides pharmaceutical compositions, and theirmethods of use, where the pharmaceutical compositions comprise aphenolic opioid prodrug that provides enzymatically-controlled releaseof a phenolic opioid, and an enzyme inhibitor that interacts with theenzyme(s) that mediates the enzymatically-controlled release of thephenolic opioid from the prodrug so as to attenuate enzymatic cleavageof the prodrug. The disclosure provides pharmaceutical compositionswhich comprise a trypsin inhibitor and a phenolic opioid prodrug thatcontains a trypsin-labile moiety that, when cleaved, facilitates releaseof phenolic opioid. Examples of phenolic opioid prodrugs and trypsininhibitors are described below.

Phenolic Opioid Prodrugs

According to certain embodiments, there is provided a phenolic opioidprodrug which provides enzymatically-controlled release of a phenolicopioid. The phenolic opioid prodrug is a corresponding compound in whichthe phenolic hydrogen atom has been substituted with a spacer leavinggroup bearing a nitrogen nucleophile that is protected with anenzymatically-cleavable moiety, the configuration of the spacer leavinggroup and nitrogen nucleophile being such that, upon enzymatic cleavageof the cleavable moiety, the nitrogen nucleophile is capable of forminga cyclic urea, liberating the compound from the spacer leaving group soas to provide a phenolic opioid.

The enzyme capable of cleaving the enzymatically-cleavable moiety may bea peptidase—the enzymatically-cleavable moiety being linked to thenucleophilic nitrogen through an amide (e.g. a peptide: —NHCO—) bond. Insome embodiments, the enzyme is a digestive enzyme of a protein.

Formulae I-VI

As shown herein, Formula I describes compounds of Formula II, in whichR¹ is (1-4C)alkyl group; R² and R³ each independently represents ahydrogen atom or a (1-4C)alkyl group; and R⁵ represents a hydrogen atom,an N-acyl group (including N-substituted acyl), a residue of an aminoacid, a dipeptide, an N-acyl derivative (including N-substituted acylderivative) of an amino acid or dipeptide.

Formula III describes compounds of Formula II, in which R¹ is(1-4C)alkyl group; R² and R³ each independently represents a hydrogenatom or a (1-4C)alkyl group; and R⁵ represents a hydrogen atom, anN-acyl group (including N-substituted acyl), a residue of an amino acid,a dipeptide, an N-acyl derivative (including N-substituted acylderivative) of an amino acid or dipeptide.

Formula IV describes compounds of Formula I, wherein “X” is replacedstructurally with certain phenolic opioids.

As also shown herein, Formula IV describes compounds of Formula V, inwhich R¹ is (1-4C)alkyl group; R² and R³ each independently represents ahydrogen atom or a (1-4C)alkyl group; and R⁵ represents a hydrogen atom,an N-acyl group (including N-substituted acyl), a residue of an aminoacid, a dipeptide, an N-acyl derivative (including N-substituted acylderivative) of an amino acid or dipeptide.

Formula VI describes componds of Formula V, in which R¹ is (1-4C)alkylgroup; R² and R³ each independently represents a hydrogen atom or a(1-4C)alkyl group; and R⁵ represents a hydrogen atom, an N-acyl group(including N-substituted acyl), a residue of an amino acid, a dipeptide,an N-acyl derivative (including N-substituted acyl derivative) of anamino acid or dipeptide.

For Formulae I-III, X represents a residue of a phenolic opioid, whereinthe hydrogen atom of the phenolic hydroxyl group is replaced by acovalent bond to —C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵).

As disclosed above, “opioid” refers to a chemical substance that exertsits pharmacological action by interaction at opioid receptors. “Phenolicopioid” refers to a subset of the opioids that contain a phenol group.For example, phenolic opioids include, but are not limited to,buprenorphine, dihydroetorphine, diprenorphine, etorphine,hydromorphone, levorphanol, morphine (and metabolites thereof),nalmefene, naloxone, N-methylnaloxone, naltrexone, N-methylnaltrexone,oxymorphone, oripavin, ketobemidone, dezocine, pentazocine, phenazocine,butorphanol, nalbuphine, meptazinol, O-desmethyltramadol, tapentado,nalorphine. The structures of the aforementioned phenolic opioids areshown below:

In certain embodiments, the phenolic opioid is oxymorphone,hydromorphone, or morphine.

Formulae I-VI are now described in more detail below.

Formula I

The compounds of formula (I) correspond with compounds disclosed in WO2007/140272 in which the nucleophilic nitrogen atom is bound to aresidue of L-arginine or L-lysine.

Examples of values for the phenolic opioid as provided in X areoxymorphone, hydromorphone and morphine.

Examples of values for R¹ are methyl and ethyl groups.

Examples of values for each of R² and R³ are hydrogen atoms.

An example of a value for n is 2.

In one embodiment, R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂.

An amino acid can be a naturally occurring amino acid. It will beappreciated that naturally occurring amino acids usually have theL-configuration.

Referring to R⁵, examples of particular values are:

for an N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, anN-aroyl group, such as N-benzoyl, or an N-piperonyl group;for an amino acid: alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine; andfor a dipeptide: a combination of any two amino acids selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

Examples of particular values for R⁵ are:

a hydrogen atom;for an N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, anN-aroyl group, such as N-benzoyl,or an N-piperonyl group; andfor a residue of an amino acid, a dipeptide, or an N-acyl derivative ofan amino acid or dipeptide: glycinyl or N-acetylglycinyl.

In one embodiment, R⁵ represents N-acetyl, glycinyl or N-acetylglycinyl,such as N-acetyl.

An example of the group represented by —C(O)—CH(R⁴)—NH(R⁵) isN-acetylarginyl.

In a particular embodiment, the compound of formula (I) is hydromorphone3-(N-methyl-N-(2-N′-acetylarginylamino)) ethylcarbamate, or apharmaceutically acceptable salt thereof. This compound is described inExample 3 of WO 2007/140272.

Formula II

The embodiments provide a compound of general formula (II):

X—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵)  (II)

or a pharmaceutically acceptable salt thereof, in which:

X represents a residue of a phenolic opioid, wherein the hydrogen atomof the phenolic hydroxyl group is replaced by a covalent bond to—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵);

R¹ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R² and R³ together with the carbon to which they are attached form acycloalkyl and substituted cycloalkyl group, or two R² or R³ groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n represents an integer from 2 to 4;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group (including N-substitutedacyl), a residue of an amino acid, a dipeptide, an N-acyl derivative(including N-substituted acyl derivative) of an amino acid or dipeptide.

In formula II, examples of values for the phenolic opioid as provided inX are oxymorphone, hydromorphone and morphine.

In formula II, R¹ can be selected from alkyl, substituted alkyl,arylalkyl, substituted arylalkyl, aryl and substituted aryl. In certaininstances, R¹ is (1-6C)alkyl. In other instances, R¹ is (1-4C)alkyl. Inother instances, R¹ is methyl or ethyl. In other instances, R¹ ismethyl. In some instances, R¹ is ethyl.

In certain instances, R¹ is substituted alkyl. In certain instances, R¹is an alkyl group substituted with carboxyl or carboxyl ester. Incertain instances, R¹ is —(CH₂)₅—COOH, —(CH₂)₅—COOCH₃, or—(CH₂)₅—COOCH₂CH₃.

In certain instances, in formula II, R¹ is arylalkyl or substitutedarylalkyl. In certain instances, in formula II, R¹ is arylalkyl. Incertain instances, R¹ is substituted arylalkyl. In certain instances, R¹is an arylalkyl group substituted with carboxyl or carboxyl ester. Incertain instances, R¹ is —(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃,or —(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10. Incertain instances, R¹ is —CH₂(C₆H₄)—COOH, —CH₂(C₆H₄)—COOCH₃, or —CH₂(C₆H₄)—COOCH₂CH₃.

In certain instances, in formula II, R¹ is aryl. In certain instances,R¹ is substituted aryl. In certain instances, R¹ is an aryl groupsubstituted with carboxyl or carboxyl ester. In certain instances, R¹ is—(C₆H₄)—COOH, —(C₆H₄)—COOCH₃, or —(C₆H₄)—COOCH₂CH₃.

In formula II, each R² can be independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl.In certain instances, R² is hydrogen or alkyl. In certain instances, R²is hydrogen. In certain instances, R¹ is alkyl. In certain instances, R²is acyl. In certain instances, R² is aminoacyl.

In formula II, each R³ can be independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl.In certain instances, R³ is hydrogen or alkyl. In certain instances, R³is hydrogen. In certain instances, R³ is alkyl. In certain instances, R³is acyl. In certain instances, R³ is aminoacyl.

In certain instances, R² and R³ are hydrogen. In certain instances, R²and R³ on the same carbon are both alkyl. In certain instances, R² andR³ on the same carbon are methyl. In certain instances, R² and R³ on thesame carbon are ethyl.

In formula II, R² and R³ together with the carbon to which they areattached can form a cycloalkyl and substituted cycloalkyl group, or twoR² or R³ groups on adjacent carbon atoms, together with the carbon atomsto which they are attached, can form a cycloalkyl or substitutedcycloalkyl group. In certain instances, R² and R³ together with thecarbon to which they are attached can form a cycloalkyl group. Thus, incertain instances, R² and R³ on the same carbon form a spirocycle. Incertain instances, R² and R³ together with the carbon to which they areattached can form a substituted cycloalkyl group. In certain instances,two R² or R³ groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, can form a cycloalkyl group. Incertain instances, two R² or R³ groups on adjacent carbon atoms,together with the carbon atoms to which they are attached, can form asubstituted cycloalkyl group.

In certain instances, one of R² and R³ is aminoacyl.

In certain instances, one of R² and R³ is aminoacyl comprisingphenylenediamine. In certain instances, one of R² and R³ is

wherein each R¹⁰ is independently selected from hydrogen, alkyl,substituted alkyl, and acyl and R¹¹ is alkyl or substituted alkyl. Incertain instances, at least one of R¹¹ is acyl. In certain instances, atleast one of R¹⁰ is alkyl or substituted alkyl. In certain instances, atleast one of R¹⁰ is hydrogen. In certain instances, both of R¹⁰ arehydrogen.

In certain instances, one of R² and R³ is

wherein R¹⁰ is hydrogen, alkyl, substituted alkyl, or acyl. In certaininstances, R¹⁰ is acyl. In certain instances, R¹⁰ is alkyl orsubstituted alkyl. In certain instances, R¹⁰ is hydrogen.

In certain instances, R² or R³ can modulate a rate of intramolecularcyclization. R² or R³ can speed up a rate of intramolecular cyclization,when compared to the corresponding molecule where R² and R³ are bothhydrogen. In certain instances, R² or R³ comprise anelectron-withdrawing group or an electron-donating group. In certaininstances, R² or R³ comprise an electron-withdrawing group. In certaininstances, R² or R³ comprise an electron-donating group.

Atoms and groups capable of functioning as electron withdrawingsubstituents are well known in the field of organic chemistry. Theyinclude electronegative atoms and groups containing electronegativeatoms. Such groups function to lower the basicity or protonation stateof a nucleophilic nitrogen in the beta position via inductive withdrawalof electron density. Such groups can also be positioned on otherpositions along the alkylene chain. Examples include halogen atoms (forexample, a fluorine atom), acyl groups (for example an alkanoyl group,an aroyl group, a carboxyl group, an alkoxycarbonyl group, anaryloxycarbonyl group or an aminocarbonyl group (such as a carbamoyl,alkylaminocarbonyl, dialkylaminocarbonyl or arylaminocarbonyl group)),an oxo (═O) substituent, a nitrile group, a nitro group, ether groups(for example an alkoxy group) and phenyl groups bearing a substituent atthe ortho position, the para position or both the ortho and the parapositions, each substituent being selected independently from a halogenatom, a fluoroalkyl group (such as trifluoromethyl), a nitro group, acyano group and a carboxyl group. Each of the electron withdrawingsubstituents can be selected independently from these.

In certain instances, —[C(R²)(R³)]_(n)— is selected from—CH(CH₂F)CH(CH₂F)—; —CH(CHF₂)CH(CHF₂)—; —CH(CF₃)CH(CF₃)—; —CH₂CH(CF₃)—;—CH₂CH(CHF₂)—; —CH₂CH(CH₂F)—; —CH₂CH(F)CH₂—; —CH₂C(F₂)CH₂—;—CH₂CH(C(O)NR²⁰R²¹)—; —CH₂CH(C(O)OR²²)—; —CH₂CH(C(O)OH)—;—CH(CH₂F)CH₂CH(CH₂F)—; —CH(CHF₂)CH₂CH(CHF₂)—; —CH(CF₃)CH₂CH(CF₃)—;—CH₂CH₂CH(CF₃)—; —CH₂CH₂CH(CHF₂)—; —CH₂CH₂CH(CH₂F)—; —CH₂CH₂CH(C(O)NR²³R²⁴)—; —CH₂CH₂CH(C(O)OR²⁵)—; and —CH₂CH₂CH(C(O)OH)—, in which R²⁰,R²¹, R²² and R²³ each independently represents hydrogen or (1-6C)alkyl,and R²⁴ and R²⁵ each independently represents (1-6C)alkyl.

In formula II, n represents an integer from 2 to 4. An example of avalue for n is 2. An example of a value for n is 3. An example of avalue for n is 4.

In formula II, in one embodiment, R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂.In another embodiment, R⁴ represents —CH₂CH₂CH₂CH₂NH₂.

An amino acid can be a naturally occurring amino acid. It will beappreciated that naturally occurring amino acids usually have theL-configuration.

In formula II, referring to R⁵, examples of particular values are: foran N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, an N-aroylgroup, such as N-benzoyl, or an N-piperonyl group;

for an amino acid: alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine; andfor a dipeptide: a combination of any two amino acids selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In formula II, examples of particular values for R⁵ are:

a hydrogen atom;for an N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, anN-aroyl group, such as N-benzoyl, or an N-piperonyl group; andfor a residue of an amino acid, a dipeptide, or an N-acyl derivative ofan amino acid or dipeptide: glycinyl or N-acetylglycinyl.

In formula II, in one embodiment, R⁵ represents N-acetyl, glycinyl orN-acetylglycinyl, such as N-acetyl.

In formula II, an example of the group represented by—C(O)—CH(R⁴)—NH(R⁵) is N-acetylarginyl or N-acetyllysinyl.

In formula II, in certain instances, R⁵ represents substituted acyl. Incertain instances, R⁵ can be malonyl or succinyl.

In formula II, in certain instances, the group represented by—C(O)—CH(R⁴)—NH(R⁵) is N-malonylarginyl, N-malonyllysinyl,N-succinylarginyl and N-succinyllysinyl.

Formula III

The embodiments provide a compound of general formula (III):

X—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵)  (III)

or a pharmaceutically acceptable salt thereof, in which:

X represents a residue of a phenolic opioid, wherein the hydrogen atomof the phenolic hydroxyl group is replaced by a covalent bond to—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵);

R¹ represents a (1-4C)alkyl group;

R² and R³ each independently represents a hydrogen atom or a (1-4C)alkylgroup;

n represents 2 or 3;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group (including N-substitutedacyl), a residue of an amino acid, a dipeptide, an N-acyl derivative(including N-substituted acyl derivative) of an amino acid or dipeptide.

In formula III, examples of values for the phenolic opioid as providedin X are oxymorphone, hydromorphone and morphine.

In formula III, examples of values for R¹ are methyl and ethyl groups.

In formula III, examples of values for each of R² and R³ are hydrogenatoms.

In formula III, an example of a value for n is 2.

In formula III, in one embodiment, R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂.In another embodiment, R⁴ represents —CH₂CH₂CH₂CH₂NH₂.

An amino acid can be a naturally occurring amino acid. It will beappreciated that naturally occurring amino acids usually have theL-configuration.

In formula III, referring to R⁵, examples of particular values are:

for an N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, anN-aroyl group, such as N-benzoyl, or an N-piperonyl group;for an amino acid: alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine; andfor a dipeptide: a combination of any two amino acids selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In formula III, examples of particular values for R⁵ are: a hydrogenatom;

for an N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, anN-aroyl group, such as N-benzoyl, or an N-piperonyl group; andfor a residue of an amino acid, a dipeptide, or an N-acyl derivative ofan amino acid or dipeptide: glycinyl or N-acetylglycinyl.

In formula III, in one embodiment, R⁵ represents N-acetyl, glycinyl orN-acetylglycinyl, such as N-acetyl.

In formula III, an example of the group represented by—C(O)—CH(R⁴)—NH(R⁵) is N-acetylarginyl or N-acetyllysinyl.

In formula III, in certain instances, R⁵ represents substituted acyl. Incertain instances, R⁵ can be malonyl or succinyl.

In formula III, in certain instances, the group represented by—C(O)—CH(R⁴)—NH(R⁵) is N-malonylarginyl, N-malonyllysinyl,N-succinylarginyl and N-succinyllysinyl.

Formula IV

The embodiments provide a compound of general formula (IV):

or a pharmaceutically acceptable salt thereof, in which:

R^(a) is hydrogen or hydroxyl;

R^(b) is oxo (═O) or hydroxyl;

the dashed line is a double bond or single bond;

R¹ represents a (1-4C)alkyl group;

R² and R³ each independently represents a hydrogen atom or a (1-4C)alkylgroup;

n represents 2 or 3;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group, or a residue of an aminoacid, a dipeptide, or an N-acyl derivative of an amino acid ordipeptide.

In formula IV, a certain example of R^(a) is hydrogen. In formula IV, acertain example of R^(a) is hydroxyl.

In formula IV, a certain example of R^(b) is oxo (═O). In formula IV, acertain example of R^(b) is hydroxyl;

In formula IV, a certain example of the dashed line is a double bond. Informula IV, a certain example of the dashed line is a single bond.

In formula IV, examples of values for R¹ are methyl and ethyl groups.

In formula IV, examples of values for each of R² and R³ are hydrogenatoms.

In formula IV, an example of a value for n is 2.

In formula IV, in one embodiment, R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂.

An amino acid can be a naturally occurring amino acid. It will beappreciated that naturally occurring amino acids usually have theL-configuration.

In formula IV, referring to R⁵, examples of particular values are: foran N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, an N-aroylgroup, such as N-benzoyl, or an N-piperonyl group;

for an amino acid: alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine; andfor a dipeptide: a combination of any two amino acids selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In formula IV, examples of particular values for R⁵ are:

a hydrogen atom;for an N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, anN-aroyl group, such as N-benzoyl, or an N-piperonyl group; andfor a residue of an amino acid, a dipeptide, or an N-acyl derivative ofan amino acid or dipeptide: glycinyl or N-acetylglycinyl.

In formula IV, in one embodiment, R⁵ represents N-acetyl, glycinyl orN-acetylglycinyl, such as N-acetyl.

In formula IV, an example of the group represented by—C(O)—CH(R⁴)—NH(R⁵) is N-acetylarginyl.

Formula V

The embodiments provide a compound of general formula (V):

or a pharmaceutically acceptable salt thereof, in which:

R^(a) is hydrogen or hydroxyl;

R^(b) is oxo (═O) or hydroxyl;

the dashed line is a double bond or single bond;

R¹ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R² and R³ together with the carbon to which they are attached form acycloalkyl and substituted cycloalkyl group, or two R² or R³ groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n represents an integer from 2 to 4;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group (including N-substitutedacyl), a residue of an amino acid, a dipeptide, an N-acyl derivative(including N-substituted acyl derivative) of an amino acid or dipeptide.

In formula V, a certain example of R^(a) is hydrogen. In formula IV, acertain example of R^(a) is hydroxyl.

In formula V, a certain example of R^(b) is oxo (═O). In formula V, acertain example of R^(b) is hydroxyl;

In formula V, a certain example of the dashed line is a double bond. Informula V, a certain example of the dashed line is a single bond.

In formula V, R¹ can be selected from alkyl, substituted alkyl,arylalkyl, substituted arylalkyl, aryl and substituted aryl. In certaininstances, R¹ is (1-6C)alkyl. In other instances, R¹ is (1-4C)alkyl. Inother instances, R¹ is methyl or ethyl. In other instances, R¹ ismethyl. In some instances, R¹ is ethyl.

In certain instances, R¹ is substituted alkyl. In certain instances, R¹is an alkyl group substituted with carboxyl or carboxyl ester. Incertain instances, R¹ is —(CH₂)₅—COOH, —(CH₂)₅—COOCH₃, or—(CH₂)₅—COOCH₂CH₃.

In certain instances, in formula V, R¹ is arylalkyl or substitutedarylalkyl. In certain instances, in formula V, R¹ is arylalkyl. Incertain instances, R¹ is substituted arylalkyl. In certain instances, R¹is an arylalkyl group substituted with carboxyl or carboxyl ester. Incertain instances, R¹ is —(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃,or —(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10. Incertain instances, R¹ is —CH₂(C₆H)—COOH, —CH₂(C₆H₄)—COOCH₃, or —CH₂(C₆H₄)—COOCH₂CH₃.

In certain instances, in formula V, R¹ is aryl. In certain instances, R¹is substituted aryl. In certain instances, R¹ is an aryl groupsubstituted with carboxyl or carboxyl ester. In certain instances, R¹ is—(C₆H₄)—COOH, —(C₆H₄)—COOCH₃, or —(C₆H₄)—COOCH₂CH₃.

In formula V, each R² can be independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl.In certain instances, R² is hydrogen or alkyl. In certain instances, R²is hydrogen. In certain instances, R¹ is alkyl. In certain instances, R²is acyl. In certain instances, R² is aminoacyl.

In formula V, each R³ can be independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl.In certain instances, R³ is hydrogen or alkyl. In certain instances, R³is hydrogen. In certain instances, R³ is alkyl. In certain instances, R³is acyl. In certain instances, R³ is aminoacyl.

In certain instances, R² and R³ are hydrogen. In certain instances, R²and R³ on the same carbon are both alkyl. In certain instances, R² andR³ on the same carbon are methyl. In certain instances, R² and R³ on thesame carbon are ethyl.

In formula V, R² and R³ together with the carbon to which they areattached can form a cycloalkyl and substituted cycloalkyl group, or twoR² or R³ groups on adjacent carbon atoms, together with the carbon atomsto which they are attached, can form a cycloalkyl or substitutedcycloalkyl group. In certain instances, R² and R³ together with thecarbon to which they are attached can form a cycloalkyl group. Thus, incertain instances, R² and R³ on the same carbon form a spirocycle. Incertain instances, R² and R³ together with the carbon to which they areattached can form a substituted cycloalkyl group. In certain instances,two R² or R³ groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, can form a cycloalkyl group. Incertain instances, two R² or R³ groups on adjacent carbon atoms,together with the carbon atoms to which they are attached, can form asubstituted cycloalkyl group.

In certain instances, one of R² and R³ is aminoacyl.

In certain instances, one of R² and R³ is aminoacyl comprisingphenylenediamine. In certain instances, one of R² and R³ is

wherein each R¹⁰ is independently selected from hydrogen, alkyl,substituted alkyl, and acyl and R¹⁰ is alkyl or substituted alkyl. Incertain instances, at least one of R¹⁰ is acyl. In certain instances, atleast one of R¹⁰ is alkyl or substituted alkyl. In certain instances, atleast one of R¹⁰ is hydrogen. In certain instances, both of R¹⁰ arehydrogen.

In certain instances, one of R² and R³ is

wherein R¹⁰ is hydrogen, alkyl, substituted alkyl, or acyl. In certaininstances, R¹⁰ is acyl. In certain instances, R¹⁰ is alkyl orsubstituted alkyl. In certain instances, R¹⁰ is hydrogen.

In certain instances, R² or R³ can modulate a rate of intramolecularcyclization. R² or R³ can speed up a rate of intramolecular cyclization,when compared to the corresponding molecule where R² and R³ are bothhydrogen. In certain instances, R² or R³ comprise anelectron-withdrawing group or an electron-donating group. In certaininstances, R² or R³ comprise an electron-withdrawing group. In certaininstances, R² or R³ comprise an electron-donating group.

Atoms and groups capable of functioning as electron withdrawingsubstituents are well known in the field of organic chemistry. Theyinclude electronegative atoms and groups containing electronegativeatoms. Such groups function to lower the basicity or protonation stateof a nucleophilic nitrogen in the beta position via inductive withdrawalof electron density. Such groups can also be positioned on otherpositions along the alkylene chain. Examples include halogen atoms (forexample, a fluorine atom), acyl groups (for example an alkanoyl group,an aroyl group, a carboxyl group, an alkoxycarbonyl group, anaryloxycarbonyl group or an aminocarbonyl group (such as a carbamoyl,alkylaminocarbonyl, dialkylaminocarbonyl or arylaminocarbonyl group)),an oxo (═O) substituent, a nitrile group, a nitro group, ether groups(for example an alkoxy group) and phenyl groups bearing a substituent atthe ortho position, the para position or both the ortho and the parapositions, each substituent being selected independently from a halogenatom, a fluoroalkyl group (such as trifluoromethyl), a nitro group, acyano group and a carboxyl group. Each of the electron withdrawingsubstituents can be selected independently from these.

In certain instances, —[C(R²)(R³)]_(n)— is selected from—CH(CH₂F)CH(CH₂F)—; —CH(CHF₂)CH(CHF₂)—; —CH(CF₃)CH(CF₃)—; —CH₂CH(CF₃)—;—CH₂CH(CHF₂)—; —CH₂CH(CH₂F)—; —CH₂CH(F)CH₂—; —CH₂C(F₂)CH₂—;—CH₂CH(C(O)NR²⁰R²¹)—; —CH₂CH(C(O)OR²²)—; —CH₂CH(C(O)OH)—;—CH(CH₂F)CH₂CH(CH₂F)—; —CH(CHF₂)CH₂CH(CHF₂)—; —CH(CF₃)CH₂CH(CF₃)—;—CH₂CH₂CH(CF₃)—; —CH₂CH₂CH(CHF₂)—; —CH₂CH₂CH(CH₂F)—; —CH₂CH₂CH(C(O)NR²³R²⁴)—; —CH₂CH₂CH(C(O)OR²⁵)—; and —CH₂CH₂CH(C(O)OH)—, in which R²⁰,R²¹, R²² and R²³ each independently represents hydrogen or (1-6C)alkyl,and R²⁴ and R²⁵ each independently represents (1-6C)alkyl.

In formula V, n represents an integer from 2 to 4. An example of a valuefor n is 2. An example of a value for n is 3. An example of a value forn is 4.

In formula V, in one embodiment, R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂. Inanother embodiment, R⁴ represents —CH₂CH₂CH₂CH₂NH₂.

An amino acid can be a naturally occurring amino acid. It will beappreciated that naturally occurring amino acids usually have theL-configuration.

In formula V, referring to R⁵, examples of particular values are: for anN-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, an N-aroylgroup, such as N-benzoyl, or an N-piperonyl group;

for an amino acid: alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine; andfor a dipeptide: a combination of any two amino acids selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In formula V, examples of particular values for R⁵ are:

a hydrogen atom;for an N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, anN-aroyl group, such as N-benzoyl, or an N-piperonyl group; andfor a residue of an amino acid, a dipeptide, or an N-acyl derivative ofan amino acid or dipeptide: glycinyl or N-acetylglycinyl.

In formula V, in one embodiment, R⁵ represents N-acetyl, glycinyl orN-acetylglycinyl, such as N-acetyl.

In formula V, an example of the group represented by —C(O)—CH(R⁴)—NH(R⁵)is N-acetylarginyl or N-acetyllysinyl.

In formula V, in certain instances, R⁵ represents substituted acyl. Incertain instances, R⁵ can be malonyl or succinyl.

In formula V, in certain instances, the group represented by—C(O)—CH(R⁴)—NH(R⁵) is N-malonylarginyl, N-malonyllysinyl,N-succinylarginyl and N-succinyllysinyl.

Formula VI

The embodiments provide a compound of general formula (VI):

or a pharmaceutically acceptable salt thereof, in which:

R^(a) is hydrogen or hydroxyl;

R^(b) is oxo (═O) or hydroxyl;

the dashed line is a double bond or single bond;

R¹ represents a (1-4C)alkyl group;

R² and R³ each independently represents a hydrogen atom or a (1-4C)alkylgroup;

n represents 2 or 3;

R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and

R⁵ represents a hydrogen atom, an N-acyl group (including N-substitutedacyl), a residue of an amino acid, a dipeptide, an N-acyl derivative(including N-substituted acyl derivative) of an amino acid or dipeptide.

In formula VI, a certain example of R^(a) is hydrogen. In formula VI, acertain example of R^(a) is hydroxyl.

In formula VI, a certain example of R^(b) is oxo (═O). In formula VI, acertain example of R^(b) is hydroxyl;

In formula VI, a certain example of the dashed line is a double bond. Informula VI, a certain example of the dashed line is a single bond.

In formula VI, examples of values for R¹ are methyl and ethyl groups.

In formula VI, examples of values for each of R² and R³ are hydrogenatoms.

In formula VI, an example of a value for n is 2.

In formula VI, in one embodiment, R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂.In another embodiment, R⁴ represents —CH₂CH₂CH₂CH₂NH₂.

An amino acid can be a naturally occurring amino acid. It will beappreciated that naturally occurring amino acids usually have theL-configuration.

In formula VI, referring to R⁵, examples of particular values are: foran N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, an N-aroylgroup, such as N-benzoyl, or an N-piperonyl group;

for an amino acid: alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine; andfor a dipeptide: a combination of any two amino acids selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In formula VI, examples of particular values for R⁵ are:

a hydrogen atom;

for an N-acyl group: an N-(1-4C)alkanoyl group, such as acetyl, anN-aroyl group, such as N-benzoyl, or an N-piperonyl group; and

for a residue of an amino acid, a dipeptide, or an N-acyl derivative ofan amino acid or dipeptide: glycinyl or N-acetylglycinyl.

In formula VI, in one embodiment, R⁵ represents N-acetyl, glycinyl orN-acetylglycinyl, such as N-acetyl.

In formula VI, an example of the group represented by—C(O)—CH(R⁴)—NH(R⁵) is N-acetylarginyl or N-acetyllysinyl.

In formula VI, in certain instances, R⁵ represents substituted acyl. Incertain instances, R⁵ can be malonyl or succinyl.

In formula VI, in certain instances, the group represented by—C(O)—CH(R⁴)—NH(R⁵) is N-malonylarginyl, N-malonyllysinyl,N-succinylarginyl and N-succinyllysinyl.

General Synthetic Procedures

Compounds of formula I are particular prodrugs described in WO2007/140272 and the synthesis of compounds of formula I are describedtherein.

The synthetic schemes and procedure in WO 2007/140272 can also be usedto synthesize compounds of formulae I-VI. The compounds described hereinmay be obtained via the routes generically illustrated in Scheme 1.

The promoieties described herein, may be prepared and attached to drugscontaining phenols by procedures known to those of skill in the art (Seee.g., Green et al., “Protective Groups in Organic Chemistry,” (Wiley,2^(nd) ed. 1991); Harrison et al., “Compendium of Synthetic OrganicMethods,” Vols. 1-8 (John Wiley and Sons, 1971-1996); “BeilsteinHandbook of Organic Chemistry,” Beilstein Institute of OrganicChemistry, Frankfurt, Germany; Feiser et al., “Reagents for OrganicSynthesis,” Volumes 1-17, (Wiley Interscience); Trost et al.,“Comprehensive Organic Synthesis,” (Pergamon Press, 1991);“Theilheimer's Synthetic Methods of Organic Chemistry,” Volumes 1-45,(Karger, 1991); March, “Advanced Organic Chemistry,” (WileyInterscience), 1991; Larock “Comprehensive Organic Transformations,”(VCH Publishers, 1989); Paquette, “Encyclopedia of Reagents for OrganicSynthesis,” (John Wiley & Sons, 1995), Bodanzsky, “Principles of PeptideSynthesis,” (Springer Verlag, 1984); Bodanzsky, “Practice of PeptideSynthesis,” (Springer Verlag, 1984). Further, starting materials may beobtained from commercial sources or via well established syntheticprocedures, supra.

Referring now to Scheme 1 and formula I, supra, where for illustrativepurposes T is NH, Y is NR¹, W is NH, p is one, R¹, R⁴, and R⁵ are aspreviously defined, X is a phenolic opioid, P is a protecting group, andM is a leaving group, compound 1-1 may be acylated with an appropriatecarboxylic acid or carboxylic acid equivalent to provide compound 1-2which then may be deprotected to yield compound 1-3. Compound 1-3 isthen reacted with an activated carbonic acid equivalent 1-4 to providecompound 1-5.

For compounds of formula II-VI, —(C(R₂)(R₃))_(n)— correspond to—(CH₂—CH₂)— portion between Y and T. Thus, for the synthesis ofcompounds of formulae II-VI, compound 1-1 would have the appropriateentities for —(C(R₂)(R₃))_(n)— to result in the synthesis of compoundsof formulae II-VI.

Trypsin Inhibitors

As used herein, the term “trypsin inhibitor” refers to any agent capableof inhibiting the action of trypsin on a substrate. The ability of anagent to inhibit trypsin can be measured using assays well known in theart. For example, in a typical assay, one unit corresponds to the amountof inhibitor that reduces the trypsin activity by one benzoyl-L-arginineethyl ester unit (BAEE-U). One BAEE-U is the amount of enzyme thatincreases the absorbance at 253 nm by 0.001 per minute at pH 7.6 and 25°C. See, for example, K. Ozawa, M. Laskowski, 1966, J. Biol. Chem. 241,3955 and Y. Birk, 1976, Meth. Enzymol. 45, 700.

There are many trypsin inhibitors known in the art, both those specificto trypsin and those that inhibit trypsin and other proteases such aschymotrypsin. Trypsin inhibitors can be derived from a variety of animalor vegetable sources: for example, soybean, corn, lima and other beans,squash, sunflower, bovine and other animal pancreas and lung, chickenand turkey egg white, soy-based infant formula, and mammalian blood.Trypsin inhibitors can also be of microbial origin: for example,antipain; see, for example, H. Umezawa, 1976, Meth. Enzymol. 45, 678. Atrypsin inhibitor can also be an arginine or lysine mimic or othersynthetic compound: for example arylguanidine, benzamidine,3,4-dichloroisocoumarin, diisopropylfluorophosphate, gabexate mesylate,or phenylmethanesulfonyl fluoride. As used herein, an arginine or lysinemimic is a compound that is capable of binding to the P¹ pocket oftrypsin and/or interfering with trypsin active site function.

In one embodiment, the trypsin inhibitor is derived from soybean.Trypsin inhibitors derived from soybean (Glycine max) are readilyavailable and are considered to be safe for human consumption. Theyinclude, but are not limited to, SBTI, which inhibits trypsin, andBowman-Birk inhibitor, which inhibits trypsin and chymotrypsin. Suchtrypsin inhibitors are available, for example from Sigma-Aldrich, St.Louis, Mo., USA.

It will be appreciated that the pharmaceutical composition according tothe embodiments may further comprise one or more other trypsininhibitors.

Small Molecule Trypsin Inhibitors

As stated above, a trypsin inhibitor can be an arginine or lysine mimicor other synthetic compound. In certain embodiments, the trypsininhibitor is an arginine mimic or a lysine mimic, wherein the argininemimic or lysine mimic is a synthetic compound.

Certain trypsin inhibitors include compounds of formula:

wherein:

Q¹ is selected from —O-Q⁴ or -Q⁴-COOH, where Q⁴ is C₁-C₄ alkyl;

Q² is N or CH; and

Q³ is aryl or substituted aryl.

Certain trypsin inhibitors include compounds of formula:

wherein:

Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where

Q⁶ is —(CH₂)_(p)—COOH;

Q⁷ is —(CH₂)_(r)—C₆H₅; and

p is an integer from one to three; and

r is an integer from one to three.

Certain trypsin inhibitors include the following:

Compound 101

(S)-ethyl 4-(5-guanidino-2- (naphthalene-2-sulfonamido)pentanoyl)piperazine- 1-carboxylate Compound 102

(S)-ethyl 4-(5-guanidino-2-(2,4,6- triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate Compound 103

(S)-ethyl 1-(5-guanidino-2- (naphthalene-2-sulfonamido)pentanoyl)piperidine- 4-carboxylate Compound 104

(S)-ethyl 1-(5-guanidino-2-(2,4,6- triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylate Compound 105

(S)-6-(4-(5-guanidino-2- (naphthalene-2-sulfonamido)pentanoyl)piperazin- 1-yl)-6-oxohexanoic acid Compound 106

4-aminobenzimidamide Compound 107

3-(4-carbamimidoylphenyl)-2- oxopropanoic acid Compound 108

(S)-5-(4- carbamimidoylbenzylamino)-5- oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido) butanamido)pentanoic acid Compound 109

6-carbamimidoylnaphthalen-2-yl 4- (diaminomethyleneamino)benzoateCompound 110

4,4′-(pentane-1,5- diylbis(oxy))dibenzimidamide

In certain embodiments, the trypsin inhibitor is SBTI, BBSI, Compound101, Compound 106, Compound 108, Compound 109, or Compound 110.

Pharmaceutical Compositions

As discussed above, the present disclosure provides pharmaceuticalcompositions which comprise a trypsin inhibitor and a phenolic opioidprodrug that contains a trypsin-labile moiety that, when cleaved,facilitates release of phenolic opioid. Examples of compositionscontaining a phenolic opioid prodrug and a trypsin inhibitors aredescribed below.

Combinations of Formulae I-VI and Trypsin Inhibitor

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general Formula (I), or apharmaceutically acceptable salt thereof.

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general Formulae (II)-(VI), or apharmaceutically acceptable salt thereof.

Certain embodiments provide for a combination of a compound of Formula Iand a trypsin inhibitor, in which the phenolic opioid of Formula I andthe trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Oxymorphone SBTI Oxymorphone BBSIOxymorphone Compound 101 Oxymorphone Compound 106 Oxymorphone Compound108 Oxymorphone Compound 109 Oxymorphone Compound 110

Certain embodiments provide for a combination of a compound of formula Iand trypsin inhibitor, in which the phenolic opioid of formula I and thetrypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Hydromorphone SBTI Hydromorphone BBSIHydromorphone Compound 101 Hydromorphone Compound 106 HydromorphoneCompound 108 Hydromorphone Compound 109 Hydromorphone Compound 110

Certain embodiments provide for a combination of a compound of formula Iand trypsin inhibitor, in which the phenolic opioid of formula I and thetrypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Morphine SBTI Morphine BBSI MorphineCompound 101 Morphine Compound 106 Morphine Compound 108 MorphineCompound 109 Morphine Compound 110

Certain embodiments provide for a combination of a compound of formula Iand trypsin inhibitor, in which the phenolic opioid of formula I and thetrypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Tapentadol SBTI Tapentadol BBSITapentadol Compound 101 Tapentadol Compound 106 Tapentadol Compound 108Tapentadol Compound 109 Tapentadol Compound 110

Certain embodiments provide for a combination of a compound of formulaII and trypsin inhibitor, in which the phenolic opioid of formula II andthe trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Oxymorphone SBTI Oxymorphone BBSIOxymorphone Compound 101 Oxymorphone Compound 106 Oxymorphone Compound108 Oxymorphone Compound 109 Oxymorphone Compound 110

Certain embodiments provide for a combination of a compound of formulaII and trypsin inhibitor, in which the phenolic opioid of formula II andthe trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Hydromorphone SBTI Hydromorphone BBSIHydromorphone Compound 101 Hydromorphone Compound 106 HydromorphoneCompound 108 Hydromorphone Compound 109 Hydromorphone Compound 110

Certain embodiments provide for a combination of a compound of formulaII and trypsin inhibitor, in which the phenolic opioid of formula II andthe trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Morphine SBTI Morphine BBSI MorphineCompound 101 Morphine Compound 106 Morphine Compound 108 MorphineCompound 109 Morphine Compound 110

Certain embodiments provide for a combination of a compound of formula11 and trypsin inhibitor, in which the phenolic opioid of formula II andthe trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Tapentadol SBTI Tapentadol BBSITapentadol Compound 101 Tapentadol Compound 106 Tapentadol Compound 108Tapentadol Compound 109 Tapentadol Compound 110

Certain embodiments provide for a combination of a compound of formulaIII and trypsin inhibitor, in which the phenolic opioid of formula IIIand the trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Oxymorphone SBTI Oxymorphone BBSIOxymorphone Compound 101 Oxymorphone Compound 106 Oxymorphone Compound108 Oxymorphone Compound 109 Oxymorphone Compound 110

Certain embodiments provide for a combination of a compound of formulaIII and trypsin inhibitor, in which the phenolic opioid of formula IIIand the trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Hydromorphone SBTI Hydromorphone BBSIHydromorphone Compound 101 Hydromorphone Compound 106 HydromorphoneCompound 108 Hydromorphone Compound 109 Hydromorphone Compound 110

Certain embodiments provide for a combination of a compound of formulaIII and trypsin inhibitor, in which the phenolic opioid of formula IIIand the trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Morphine SBTI Morphine BBSI MorphineCompound 101 Morphine Compound 106 Morphine Compound 108 MorphineCompound 109 Morphine Compound 110

Certain embodiments provide for a combination of a compound of formulaIII and trypsin inhibitor, in which the phenolic opioid of formula IIIand the trypsin inhibitor are shown in the following table.

Phenolic opioid Trypsin inhibitor Tapentadol SBTI Tapentadol BBSITapentadol Compound 101 Tapentadol Compound 106 Tapentadol Compound 108Tapentadol Compound 109 Tapentadol Compound 110

Certain embodiments provide for a combination of Compound 1 and trypsininhibitor, in which the trypsin inhibitor is shown in the followingtable.

Compound Trypsin inhibitor Compound 1 SBTI Compound 1 BBSI Compound 1Compound 101 Compound 1 Compound 106 Compound 1 Compound 108 Compound 1Compound 109 Compound 1 Compound 110

Certain embodiments provide for a combination of Compound 2 and trypsininhibitor, in which the trypsin inhibitor is shown in the followingtable.

Compound Trypsin inhibitor Compound 2 SBTI Compound 2 BBSI Compound 2Compound 101 Compound 2 Compound 106 Compound 2 Compound 108 Compound 2Compound 109 Compound 2 Compound 110

Certain embodiments provide for a combination of Compound 3 and trypsininhibitor, in which the trypsin inhibitor is shown in the followingtable.

Compound Trypsin inhibitor Compound 3 SBTI Compound 3 BBSI Compound 3Compound 101 Compound 3 Compound 106 Compound 3 Compound 108 Compound 3Compound 109 Compound 3 Compound 110

Certain embodiments provide for a combination of Compound 4 and trypsininhibitor, in which the trypsin inhibitor is shown in the followingtable.

Compound Trypsin inhibitor Compound 4 SBTI Compound 4 BBSI Compound 4Compound 101 Compound 4 Compound 106 Compound 4 Compound 108 Compound 4Compound 109 Compound 4 Compound 110

It is to be appreciated that the invention also includes inhibitors ofother enzymes involved in protein assimilation that can be used incombination with a prodrug having a formula or any of I-VI comprising anamino acid of alanine, arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine.

The pharmaceutical composition according to the embodiments can furthercomprise a pharmaceutically acceptable carrier. The composition isconveniently formulated in a form suitable for oral (including buccaland sublingual) administration, for example as a tablet, capsule, thinfilm, powder, suspension, solution, syrup, dispersion or emulsion. Thecomposition can contain components conventional in pharmaceuticalpreparations, e.g. one or more carriers, binders, lubricants, excipients(e.g., to impart controlled release characteristics), pH modifiers,sweeteners, bulking agents, coloring agents or further active agents.

The pharmaceutical composition according to the embodiments is useful,for example, in the treatment of a patient suffering from, or at risk ofsuffering from pain.

The patient can be a human or a non-human animal, for example acompanion animal such as a cat, dog or horse.

Methods of Administration

The amount of compound of formulae (I)-(VI) to be administered to apatient to be effective (i.e. to provide blood levels of phenolic opioidsufficient to be effective in the treatment or prophylaxis of pain) willdepend upon the bioavailability of the particular compound, thesusceptibility of the particular compound to enzyme activation in thegut, the amount and potency of trypsin inhibitor present in thecomposition, as well as other factors, such as the species, age, weight,sex, and condition of the patient, manner of administration and judgmentof the prescribing physician. In general, the dose can be in the rangeof from 0.01 to 20 milligrams per kilogram (mg/kg) body weight. Forexample, a compound comprising a residue of hydromorphone can beadministered at a dose equivalent to administering free hydromorphone inthe range of from 0.02 to 0.5 mg/kg body weight or 0.01 to 10 mg/kg bodyweight or 0.01 to 2 mg/kg body weight. In one embodiment, the compoundcan be administered at a dose such that the level of phenolic opioidachieved in the blood is in the range of from 0.5 ng/ml to 10 ng/ml.

The amount of a trypsin inhibitor to be administered to the patient tobe effective (i.e. to attenuate release of phenolic opioid whenadministration of a compound of formulae (I)-(VI) alone would lead tooverexposure of the phenolic opioid) will depend upon the effective doseof the particular compound and the potency of the particular inhibitor,as well as other factors, such as the species, age, weight, sex andcondition of the patient, manner of administration and judgment of theprescribing physician. In general, the dose of inhibitor can be in therange of from 0.05 to 50 mg per mg of compound of formulae (I)-(VI). Ina certain embodiment, the dose of inhibitor can be in the range of from0.001 to 50 mg per mg of compound of formulae (I)-(VI).

Therapeutic Applications

In another aspect, the embodiments provide a pharmaceutical compositionas described hereinabove for use in the treatment of pain.

The present disclosure provides use of a phenolic opioid prodrug and atrypsin inhibitor in the treatment of pain.

The present disclosure provides use of a phenolic opioid prodrug and atrypsin inhibitor in the manufacture of a medicament for treatment ofpain.

In another aspect, the embodiments provides method of treating pain in apatient requiring treatment, which comprises administering an effectiveamount of a pharmaceutical composition as described hereinabove.

Thwarting Tamerping by Trypsin Mediated Release of Phenolic Opioid fromProdrugs

The disclosure provides for a composition comprising a compound offormulae I-VI and a trypsin inhibitor that reduces drug abuse potential.A trypsin inhibitor can thwart the ability of a user to apply trypsin toeffect the release of a phenolic opioid from the phenolic opioid prodrugin vitro. For example, if an abuser attempts to incubate trypsin with acomposition of the embodiments that includes a phenolic opioid prodrugand a trypsin inhibitor, the trypsin inhibitor can reduce the action ofthe added trypsin, thereby thwarting attempts to release phenolic opioidfor purposes of abuse.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Celsius, and pressure is ator near atmospheric. Standard abbreviations may be used.

Example 1 Oral Administration of Compound 1 and SBTI Trypsin Inhibitorto Rats

Hydromorphone 3-(N-methyl-N-(2-N′-acetylarginylamino)) ethylcarbamate(which can be produced as described in PCT International Publication No.WO 2007/140272, published 6 Dec. 2007, Example 3, hereinafter referredto as Compound 1) and SBTI (trypsin inhibitor from Glycine max (soybean)(Catalog No. 93620, ˜10,000 units per mg, Sigma-Aldrich) were eachdissolved in saline.

Saline solutions of Compound 1 and SBTI were dosed as indicated in Table1 via oral gavage into jugular vein-cannulated male Sprague Dawley ratsthat had been fasted for 16-18 hours (hr) prior to oral dosing; 4 ratswere dosed per group. When SBTI was dosed, it was administered 5 minutes(min) prior to Compound 1. At specified time points, blood samples weredrawn, quenched into methanol, centrifuged at 14,000 rpm @ 4° C., andstored at −80° C. until analysis by high performance liquidchromatography/mass spectrometry (HPLC/MS).

Table 1 indicates the results for rats administered a constant amount ofCompound 1 and variable amounts of SBTI. Results are reported as maximumblood concentration of hydromorphone (average±standard deviation) foreach group of 4 rats.

TABLE 1 Maximum concentration (Cmax) of hydromorphone in rat bloodCompound 1 SBTI Cmax (ng/ml (mg/kg) (mg/kg) HM) 20 0 16.5 ± 5.3  20 108.9 ± 1.8 20 100 6.0 ± 4.0 20 500 <5 20 1000 <5

-   -   Lower limit of quantitation was 1 nanogram per milliliter        (ng/ml) for the first group and 5 ng/ml for the other groups.        The results in Table 1 indicate that SBTI attenuates Compound        1's ability to release hydromorphone in a dose-dependent manner        that can approach approximately 100% attenuation at higher SBTI        concentrations.

Data obtained from the rats represented in Table 1 are also provided inFIG. 1 which compares mean blood concentrations (±standard deviations)over time of hydromorphone following PO administration to rats of 20mg/kg Compound 1 (a) alone (solid line with closed circle symbols), (b)with 10 mg/kg SBTI (dashed line with open square symbols), (c) with 100mg/kg SBTI (dotted line with open triangle symbols), (d) with 500 mg/kgSBTI (solid line with X symbols) or (e) with 1000 mg/kg SBTI (solid linewith closed square symbols). The results in FIG. 1 indicate that SBTIattenuation of Compound 1's ability to release hydromorphone suppressesCmax and delays Tmax of such hydromorphone release into the blood ofrats administered Compound 1 and 10, 100, 500 or 1000 mg/kg SBTI.

Example 2 Oral Administration of Compound 1 and SBTI Trypsin Inhibitor,in the Presence of Ovalbumin, to Rats

In an effort to understand the role of SBTI, ovalbumin was used as anon-trypsin inhibitor protein control. Albumin from chicken egg white(ovalbumin) (Catalog No. A7641, Grade VII, lyophilized powder,Sigma-Aldrich) was dissolved in saline.

Saline solutions of Compound 1 and SBTI (as described in Example 1) andof ovalbumin were combined and dosed as indicated in Table 2 via oralgavage into jugular vein-cannulated male Sprague Dawley rats (4 pergroup) that had been fasted for 16-18 hr prior to oral dosing. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 microliters(μl) plasma transferred from each sample into a fresh tube containing 1μl of formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored until analysis by HPLC/MS.

Table 2 indicates the results for rats administered Compound 1 with orwithout various amounts of ovalbumin (OVA) and/or SBTI as indicated.Results are reported as maximum plasma concentration of hydromorphone(average±standard deviation) for each group of 4 rats.

TABLE 2 Maximum concentration (Cmax) of hydromorphone in rat plasmaCompound 1 (mg/kg) OVA (mg/kg) SBTI (mg/kg) Cmax (ng/ml HM) 20 0 0 13.3± 3.7 20 20 0 11.0 ± 5.4 20 100 0  9.7 ± 3.1 20 500 0 11.6 ± 2.5 20 10000 10.3 ± 3.5 20 500 500  1.9 ± 0.9

-   -   Lower limit of quantitation was 12.5 picograms/ml (pg/ml) for        the first group, 25 pg/ml for the last group, and 100 pg/ml for        the other groups.        The results in Table 2 indicate that ovalbumin does not        significantly affect Compound 1's ability to release        hydromorphone or SBTI's ability to attenuate such release.

Data obtained from the rats represented in rows 1, 4 and 6 of Table 2are also provided in FIG. 2 which compares mean plasma concentrations(±standard deviations) over time of hydromorphone following POadministration to rats of 20 mg/kg Compound 1 (a) alone (solid line withcircle symbols), (b) with 500 mg/kg OVA (dashed line with trianglesymbols) or (c) with 500 mg/kg OVA and 500 mg/kg SBTI (dotted line withsquare symbols). The results in FIG. 2 indicate that SBTI attenuation ofCompound 1's ability to release hydromorphone suppresses Cmax and delaysTmax of such hydromorphone in plasma, even in the presence of ovalbuminRats administered 20 mg/kg Compound 1 with 500 mg/kg OVA and 500 mg/kgSBTI displayed a plasma Tmax of 8.0 hr, whereas rats administered 20mg/kg Compound 1 alone displayed a plasma Tmax of 2.3 hr. The results inTable 2 and FIG. 2 also indicate that SBTI is acting specifically byinhibiting trypsin rather than in a non-specific manner.

Example 3 Oral Administration of Compound 1 and BBSI Inhibitor to Rats

Compound 1 and BBSI (Bowman-Birk trypsin-chymotrypsin inhibitor fromGlycine max (soybean), Catalog No. T9777, Sigma-Aldrich) were eachdissolved in saline.

Saline solutions of Compound 1 and BBSI were dosed as indicated in Table3. Dosing, sampling and analysis procedures were as described in Example1.

Table 3 indicates the results for rats administered Compound 1 with orwithout BBSI. Results are reported as maximum blood concentration ofhydromorphone (average±standard deviation) for each group of 4 rats(n=4) as well as for 3 of the 4 rats administered Compound 1 and BBSI(n=3).

TABLE 3 Maximum concentration (Cmax) of hydromorphone in rat bloodCompound 1 BBSI Cmax Number of (mg/kg) (mg/kg) (ng/ml HM) Rats (n) 20 016.5 ± 5.3 n = 4 20 100 10.6 ± 5.9 n = 3 20 100  18.7 ± 17.0 n = 4

-   -   Lower limit of quantitation was 1 ng/ml for both groups. Cmax of        rat not included in n=3 analysis was 43 ng/ml; range of other        rats was 6.8-17 ng/ml.        The results in Table 3 indicate that BB SI can attenuate        Compound 1's ability to release hydromorphone.

Data obtained from the individual rats represented in Table 3, rows 1and 3 are provided in FIG. 3 which compares individual bloodconcentrations over time of hydromorphone following PO administration torats of 20 mg/kg Compound 1 (a) alone (solid lines) or (b) with 100mg/kg BBSI (dotted lines). The results in FIG. 3 indicate that BBSIattenuation of Compound 1's ability to release hydromorphone suppressesCmax and delays Tmax of such hydromorphone in blood, at least for 3 ofthe 4 rats administered Compound 1 and BBSI.

Example 4 Oral Administration of Compound 2 and SBTI Trypsin Inhibitorto Rats

Saline solutions of Compound 2 (which can be prepared as described inExample 11) and SBTI (which can be prepared as described in Example 1)were dosed as indicated in Table 4 via oral gavage into jugularvein-cannulated male Sprague Dawley rats (4 per group) that had beenfasted for 16-18 hr prior to oral dosing. When SBTI was dosed, it wasadministered 5 min prior to Compound 4. At specified time points, bloodsamples were drawn, processed and analyzed as described in Example 2.

Table 4 and FIG. 4 provide results for rats administered 20 mg/kg ofCompound 2 with or without 500 mg/kg of SBTI as indicated. Results inTable 4 are reported, for each group of 4 rats, as (a) maximum plasmaconcentration (Cmax) of hydromorphone (HM) (average±standard deviation)and (b) time after administration of Compound 2, with or without SBTI,to reach maximum hydromorphone concentration (Tmax).

TABLE 4 Cmax and Tmax of hydromorphone in rat plasma Compound 2 SBTICmax (ng/ml (mg/kg) (mg/kg) HM) Tmax (hr) 20 0 14.2 ± 2.6 2.0 20 500 7.3 ± 3.5 3.5

-   -   Lower limit of quantitation was 0.0125 ng/ml for both groups.

FIG. 4 compares mean plasma concentrations (±standard deviations) overtime of hydromorphone release following PO administration of 20 mg/kgCompound 2 alone (solid line) or with 500 mg/kg SBTI (dotted line) torats.

The results in Table 4 and FIG. 4 indicate that SBTI attenuates Compound2's ability to release hydromorphone, both with respect to suppressingCmax and delaying Tmax.

Example 5 Oral administration of Compound 3 and SBTI trypsin inhibitorto rats

Saline solutions of Compound 3 (which can be prepared as described inExample 12) and SBTI were dosed as indicated in Table 5. Dosing,sampling and analysis procedures were as described in Example 4.

Table 5 and FIG. 5 provide results for rats administered 20 mg/kg ofCompound 3 with or without 500 mg/kg of SBTI as indicated. Results inTable 5 are reported as Cmax and Tmax of hydromorphone in plasma foreach group of 4 rats.

TABLE 5 Cmax and Tmax of hydromorphone in rat plasma Compound 3 SBTICmax (ng/ml (mg/kg) (mg/kg) HM) Tmax (hr) 20 0 9.0 ± 3.1 2.3 20 500 2.3± 1.7 7.3

-   -   Lower limit of quantitation was 0.100 ng/ml for both groups.

FIG. 5 compares mean plasma concentrations (±standard deviations) overtime of hydromorphone release following PO administration of 20 mg/kgCompound 3 alone (solid line) or with 500 mg/kg SBTI (dotted line) torats.

The results in Table 5 and FIG. 5 indicate that SBTI attenuates Compound3's ability to release hydromorphone, both with respect to suppressingCmax and delaying Tmax.

Example 6 Oral Administration of Compound 4 and SBTI Trypsin Inhibitorto Rats

Saline solutions of Compound 4 (which can be prepared as described inExample 13) and SBTI were dosed as indicated in Table 6. Dosing,sampling and analysis procedures were as described in Example 4, exceptthat Compound 4 without inhibitor was administered to 7 rats.

Table 6 and FIG. 6 provide results for rats administered 20 mg/kg ofCompound 4 with or without 500 mg/kg of SBTI as indicated. Results inTable 6 are reported as Cmax and Tmax of hydromorphone in plasma foreach group of 4 rats.

TABLE 6 Cmax and Tmax of HM in rat plasma Compound 4 SBTI Cmax (ng/mlTmax Number of rats (mg/kg) (mg/kg) HM) (hr) (n) 20 0 7.7 ± 2.3 2.3 7 20500 7.5 ± 2.1 6.5 4

-   -   Lower limit of quantitation was 0.500 ng/ml for both groups.

FIG. 6 compares mean plasma concentrations (±standard deviations) overtime of hydromorphone release following PO administration of 20 mg/kgCompound 4 alone (solid line) or with 500 mg/kg SBTI (dotted line) torats.

The results in Table 6 and FIG. 6 indicate that SBTI attenuates Compound4's ability to release hydromorphone, at least with respect to delayingTmax.

Example 7 In Vitro 1050 Data

Several candidate trypsin inhibitors, namely Compounds 101-105, 107 and108 were produced as described in Examples 14-18, 19 and 20,respectively. Compound 106 (also known as 4-aminobenzamidine), Compound109 (also known as nafamostat mesylate) and Compound 110 (also known aspentamidine isethionate salt) are available from Sigma-Aldrich (St.Louis, Mo.).

The half maximal inhibitory concentration (IC50 or IC₅₀) values of eachof Compounds 101-110 as well as of SBTI and BBSI were determined using amodified trypsin assay as described by Bergmeyer, H U et al, 1974,Methods of Enzymatic Analysis Volume 1, 2^(nd) edition, 515-516,Bergmeyer, H U, ed., Academic Press, Inc. New York, N.Y.

Table 7 indicates the IC50 values for each of the designated trypsininhibitors.

TABLE 7 IC50 values of certain trypsin inhibitors Compound IC50 value101 2.0E−5 102 7.5E−5 103 2.3E−5 104 2.7E−5 105 4.1E−5 106 2.4E−5 1071.9E−6 108 8.8E−7 109 9.1E−7 110 1.8E−5 SBTI 2.7E−7 BBSI 3.8E−7

The results of Table 7 indicate that each of Compounds 101-110 exhibitstrypsin inhibition activity.

Example 8 Effect of Trypsin Inhibitors on In Vitro Trypsin-MediatedTrypsin Release of Hydromorphone from Compound 4

Compound 4 (which can be produced as described in Example 13) wasincubated with trypsin from bovine pancreas (Catalog No. T8003, Type I,˜10,000 BAEE units/mg protein, Sigma-Aldrich) in the absence or presenceof one of the following trypsin inhibitors: SBTI, Compound 107, Compound108 or Compound 109. When a trypsin inhibitor was part of the incubationmixture, Compound 4 was added 5 min after the other incubationcomponents. The reactions were conducted at 37° C. for 24 hr. Sampleswere collected at specified time points, transferred into 0.5% formicacid in acetonitrile to stop trypsin activity and stored at less than−70° C. until analysis by LC-MS/MS.

The final incubation mixtures consisted of the following components:

Incubation Components Compound Inhibitor Tris pH 8 CaCl₂ TrypsinCompound 4 Control 0 40 mM 22.5 mM 0.0228 mg/mL 0.51 mg/ml 107 1.67mg/mL 20 mM 22.5 mM 0.0228 mg/mL 0.51 mg/ml 108 1.67 mg/mL 20 mM 22.5 mM0.0228 mg/mL 0.51 mg/ml 109 1.67 mg/mL 20 mM 22.5 mM 0.0228 mg/mL 0.51mg/ml SBTI  10 mg/mL 20 mM 22.5 mM 0.0228 mg/mL 0.51 mg/ml

FIGS. 7A and 7B indicate the results of exposure of 0.51 mg/ml Compound4 to 22.8 ng/ml trypsin in the absence of any trypsin inhibitor (diamondsymbols) or in the presence of 10 mg/ml SBTI (circle symbols), 1.67mg/ml Compound 107 (upward-pointing triangle symbols), 1.67 mg/mlCompound 108 (square symbols) or 1.67 mg/ml Compound 109(downward-pointing triangles symbols). Specifically, FIG. 7A depicts thedisappearance of Compound 4, and FIG. 7B depicts the appearance ofhydromorphone, over time under these conditions.

The results in FIGS. 7A and 7B indicate that a trypsin inhibitor of theembodiments can thwart the ability of a user to apply trypsin to effectthe release of hydromorphone from Compound 4.

Example 9 Oral Administration of Compound 3 and Compound 101 TrypsinInhibitor to Rats

Saline solutions of Compound 3 (which can be prepared as described inExample 12) and Compound 101 (prepared as described in Example 14) weredosed as indicated in Table 8. Dosing, sampling and analysis procedureswere as described in Example 4, except that Compound 3 and Compound 101were combined for dosing.

Table 8 and FIG. 8 provide results for rats administered 20 mg/kg ofCompound 3 with or without 10 mg/kg of Compound 101 as indicated.Results in Table 8 are reported as Cmax and Tmax of hydromorphone inplasma for each group of 4 rats.

TABLE 8 Cmax and Tmax of HM in rat plasma Compound 3 Compound Cmax(ng/ml (mg/kg) 101 (mg/kg) HM) Tmax (hr) 20 0 9.0 ± 3.1 2.3 20 10 3.8 ±2.9 3.5

-   -   Lower limit of quantitation was 0.100 ng/ml for the first group        and 0.500 ng/ml for the second group.

FIG. 8 compares mean plasma concentrations (±standard deviations) overtime of hydromorphone release following PO administration of 20 mg/kgCompound 3 alone (solid line) or with 10 mg/kg Compound 101 (dottedline) to rats.

The results in Table 8 and FIG. 8 indicate that Compound 101 attenuatesCompound 3's ability to release hydromorphone, both with respect tosuppressing Cmax and delaying Tmax.

Example 10 Oral Administration of Compound 4 and Compound 101 TrypsinInhibitor to Rats

Saline solutions of Compound 4 (which can be prepared as described inExample 13) and Compound 101 (prepared as described in Example 14) weredosed as indicated in Table 9. Dosing, sampling and analysis procedureswere as described in Example 4, except that Compound 4 and Compound 101were combined for dosing, and Compound 4 without inhibitor wasadministered to 7 rats.

Table 9 and FIG. 9 provide results for rats administered 20 mg/kg ofCompound 4 with or without 10 mg/kg of Compound 101 as indicated.Results in Table 9 are reported as Cmax and Tmax of hydromorphone inplasma for each group of 4 rats.

TABLE 9 Cmax and Tmax of HM in rat plasma Compound 4 Compound Cmax(ng/ml Tmax Number of (mg/kg) 101 (mg/kg) HM) (hr) rats (n) 20 0 7.7 ±2.3 2.3 7 20 10 4.8 ± 1.4 6.0 4

-   -   Lower limit of quantitation was 0.500 ng/ml for both groups.

FIG. 9 compares mean plasma concentrations (±standard deviations) overtime of hydromorphone release following PO administration of 20 mg/kgCompound 4 alone (solid line) or with 10 mg/kg Compound 101 (dottedline) to rats.

The results in Table 9 and FIG. 9 indicate that Compound 101 attenuatesCompound 4's ability to release hydromorphone, both with respect tosuppressing Cmax and delaying Tmax.

Example 11 Synthesis of[2-((S)-2-amino-5-guanidino-pentanoylamino)-ethyl]-methyl-carbamic acidhydromorphyl ester (Compound 2)

Preparation 1 Synthesis of2,2,2-trifluoro-N-(2-methylamino-ethyl)-acetamide (A)

A solution of N-methylethylenediamine (27.0 g, 364.0 mmol) and ethyltrifluoroacetate (96.6 ml, 838.0 mmol) in a mixture of acetonitrile (350ml) and water (7.8 ml, 436 mmol) was refluxed overnight with stirring.Next the solvents were evaporated in vacuo. Residue was re-evaporatedwith isopropanol (3×100 ml). Residue was dissolved in dichloromethane(500 ml) and left overnight at room temperature. The formed crystalswere filtered, washed with dichloromethane and dried in vacuo to providecompound A (96.8 g, 94%) as white solid powder.

Preparation 2 Synthesis of{methyl-[2-(2,2,2-trifluoro-acetylamino)-ethyl]carbamic acid benzylester (B)

A solution of compound A (96.8 g, 340.7 mmol) and DIEA (59.3 ml, 340.7mmol) in THF (350 ml) was cooled to −5° C., followed by addition of asolution of N-(benzyloxycarbonyl)succinimide (84.0 g, 337.3 mmol) in THF(150 ml) dropwise over the period of 20 min. The temperature of reactionmixture was raised to room temperature and stirring was continued for anadditional 30 min, followed by the solvents being evaporated.

The resultant residue was dissolved in EtOAc (600 ml). EtOAc wasextracted with 5% aq. NaHCO₃ (2×150 ml) and brine (150 ml). The organiclayer was separated and evaporated to provide compound B as yellowishoil (103.0 g, 340.7 mmol). LC-MS [1\4+H] 305.3 (C₁₃H₁₅F₃N₂O₃+H, calc:305.3). Compound B was used without further purification.

Preparation 3 Synthesis of (2-amino-ethyl)-methyl-carbamic acid benzylester (C)

To a solution of compound B (103.0 g, 340.7 mmol) in MeOH (1200 ml) wasadded a solution of LiOH (16.4 g, 681.4 mmol) in water (120 ml). Thereaction mixture was stirred at room temperature for 3 h. Solvents wereevaporated to ¾ of initial volume followed by dilution with water (400ml). Solution was extracted with EtOAc (2×300 ml). The organic layer waswashed with brine (200 ml), dried over MgSO₄ and evaporated in vacuo.The resultant residue was dissolved in ether (300 ml) and treated with 2N HCl/ether (200 ml). The formed precipitate was filtered, washed withether and dried in vacuo to provide hydrochloric salt of compound C(54.5 g, 261.2 mmol) as white solid. LC-MS [M+H] 209.5 (C₁₁H₁₆N₂O₂+H,calc: 209.3).

Preparation 4 Synthesis of{(S)-4-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-1-[2-(benzyloxycarbonyl-methyl-amino)-ethylcarbamoyl]-butyl}-carbamic acid tert-butyl ester (D)

A solution of Boc-Arg(Pbf)-OH (3.33 g, 6.32 mmol), HATU (2.88 g, 7.58mmol) and DIEA (7.4 ml, 31.6 mmol) in DMF (40 ml) was maintained at roomtemperature for 20 min, followed by the addition of compound Chydrochloride (1.45 g, 6.95 mmol). Stirring was continued for additional1 h. The reaction mixture was diluted with EtOAc (500 ml) and extractedwith water (3×75 ml) and brine (75 ml). The organic layer was dried overMgSO₄ and then evaporated to provide compound D (4.14 g, 5.77 mmol) asyellowish amorphous solid. LC-MS [M+H] 717.6 (C₃₅H₅₂N₆O₈S+H, calc:717.9).

Preparation 5 Synthesis of(S)-2-amino-5-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-pentanoicacid (2-methylamino-ethyl)-amide (E)

Compound D (4.14 g, 5.77 mmol) and AcOH (330 μl, 5.77 mmol) wasdissolved in methanol (40 ml) followed by the addition of Pd/C (5% wt,880 mg) suspension in water (5 ml). The reaction mixture was subjectedto hydrogenation (Parr apparatus, 75 psi) at room temperature for 2.5 h.The catalyst was filtered over a pad of Celite on sintered glass funneland washed with methanol. Filtrate was evaporated in vacuo to providecompound E (1.96 g, 3.2 mmol) as yellowish amorphous solid. LC-MS [M+H]483.2 (C₂₂H₃₈N₆O₄S+H, calc: 483.2).

Preparation 6 Synthesis of{(S)-4-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-1-[2-(hydromorphylcarbonyl-methyl-amino)-ethylcarbamoyl]-butyl}-carbamic acid tert-butyl ester (F)

A suspension of hydromorphone hydrochloride (332 mg, 1.03 mmol) and DIEA(179 μl, 1.03 mmol) in chloroform (4 ml) was sonicated in an ultrasonicbath at room temperature for 1 h. This was followed by the addition of4-nitrophenyl chloroformate (162 mg, 0.80 mmol). The reaction mixturewas sonicated in an ultrasonic bath at room temperature for additional 1h, followed by the addition of solution of compound E (400 mg, 0.67mmol) and 1-hydroxybenzo-triazole (154 mg, 1.14 mmol) in DMF (4 ml). Thereaction mixture was stirred overnight (˜18 h) at room temperature,followed by the solvents being evaporated in vacuo. The residue wasdissolved in MeOH (5 ml) and precipitated with addition of ether (500ml). The formed precipitate was filtered and dried in vacuo to providecompound F (520 mg, yield exceeded quantitative) as off-white solid.LC-MS [M+H] 894.6 (C₄₅H₆₃N₇O₁₀S+H, calc: 894.9).

Synthesis of[2-((S)-2-amino-5-guanidino-pentanoylamino)-ethyl]-methyl-carbamic acidhydromorphyl ester (Compound 2)

Compound F (679 mg, 0.76 mmol) was dissolved in the mixture of 5%m-cresol/TFA (10 ml). The reaction mixture was maintained at roomtemperature for 1 h, followed by the dilution with ether (500 ml).Formed precipitate was filtered, washed with ether (100 ml) and dried invacuo to provide crude compound 2 (441 mg, yield exceeded quantitative)as off-white solid. LC-MS [M+H] 542.4 (C₂₇H₃₉N₇O₅+H, calc: 542).

Crude compound 2 was dissolved in water (10 ml) and subjected topreparative reverse phase HPLC purification. [Nanosyn-Pack Microsorb(100-10)C-18 column (50×300 mm); flow rate=100 ml/min; injection volume10 ml; mobile phase A: 100% water, 0.1% TFA; mobile phase B: 100%acetonitrile, 0.1% TFA; isocratic elution at 0% B in 5 min, gradientelution to 6% B in 6 min, isocratic elution at 6% B in 23 min, gradientelution from 6% B to 55% B in 66 min; detection at 254 nm]. Fractionscontaining the desired compound were combined and concentrated in vacuo.Residue was dissolved in i-PrOH (20 ml) and evaporated in vacuo(procedure was repeated twice). Residue was dissolved in i-PrOH (2 ml)and treated with 2 N HCl/ether (100 ml, 200 mmol) to provide thehydrochloride salt of Compound 2 (80 mg, 17% yield, 98% purity) as whitesolid. LC-MS [M+H] 542.0 (C₂₇H₃₉N₇O₅+H, calc: 542.9). Retention time*:2.04 min

*-[Chromolith SpeedRod RP-18e C18 column (4.6×50 mm); flow rate 1.5ml/min; mobile phase A: 0.1% TFA/water; mobile phase B 0.1% TFA/ACN;gradient elution from 5% B to 100% B over 9.6 min, detection 254 nm]

Example 12 Synthesis of (S)-2-Acetylamino-6-amino-hexanoic acid(2-methylamino-ethyl)-amide hydromorphone ester (Compound 3)

Preparation 7 Synthesis of{(S)-1-[2-(Benzyloxycarbonyl-methyl-amino)-ethylcarbamoyl]-5-tert-butoxycarbonylamino-pentyl}-carbamicacid 9H-fluoren-9-ylmethyl ester (G)

To a solution of Fmoc-Lys(Boc)-OH (2.0 g, 4.26 mmol) in DMF (50 mL) wasadded DIEA (2.38 mL, 13.65 mmol) and stirred for 15 min at roomtemperature. The reaction mixture was then cooled to ˜5° C., followed byaddition of HATU (1.95 g, 5.12 mmol) added in portions and stirred for30 min. The CBZ-diamine (1.05 g, 4.26 mmol) was added to the reactionmixture and stirred at room temperature for 2 h. The reaction mixturewas diluted with EtOAc (250 mL), washed with water (250 mL) and brine(250 mL). The organic layer was separated, dried over Na₂SO₄, andremoval of the solvent in vacuo afforded compound G (2.3 g, 82%). LC-MS[M+H] 659.6 (C₃H₄₆N₄O₇+H, calc: 659.7).

Preparation 8 Synthesis of{(S)-5-Amino-5-[2-(benzyloxycarbonyl-methyl-amino)-ethylcarbamoyl]-pentyl}-carbamicacid tert-butyl ester (H)

To a solution of compound G (2.3 g, 3.49 mmol) in EtOAC (50 ml) wasadded piperidine (0.34 mL, 3.49 mmol). The reaction mixture was stirredfor 18 h at room temperature and then the solvents were removed invacuo. The residue was dissolved in a minimum amount of EtOAc, and thenwas precipitated with Et₂O. Precipitate was filtered off and washed withEt₂O and dried to afford compound H (1.4 g, 94%). LC-MS [M+H] 437.6(C₂₂H₃₆N₄O₅+H, calc: 437.5).

Preparation 9 Synthesis of{(S)-5-Acetylamino-5-[2-(benzyloxycarbonyl-methyl-amino)-ethylcarbamoyl]-pentyl}-carbamic acid isopropyl ester (I)

To a solution of compound H (1.4 g, 3.21 mmol) in CHCl₃ (10 mL) at roomtemperature was added DIEA (2.6 mL, 15 mmol) followed by Ac₂O (0.85 mL,9.0 mmol). The reaction mixture was stirred at room temperature for 2 h.Solvents were removed in vacuo and then the residue was dissolved indichloromethane (100 mL). The organic layer was washed with 10% citricacid (75 mL), saturated NaHCO₃ (75 mL) and brine (75 mL). The organiclayer was separated, dried over Na₂SO₄ and solvent removed in vacuo toafford compound I (1.45 g, 99%). LC-MS [M+H] 479.5 (C₂₄H₃₈N₄O₆+H, calc:479.5).

Preparation 10 Synthesis of[(S)-5-Acetylamino-5-(2-methylamino-ethylcarbamoyl)-pentyl]-carbamicacid tert-butyl ester (J)

To a solution of compound I (1.4 g, 3.00 mmol) in MeOH (40 mL) was added5% Pd/C (300 mg). This reaction mixture was subjected to hydrogenationat 70 psi for 2 h. Next, the reaction mixture was filtered through acelite pad, MeOH was removed in a rotary evaporator to afford compound J(1.02 g, 98%). LC-MS [M+H] 344.9 (C₁₆H₃₂N₄O₄+H, calc: 345.4).

Preparation 11[(S)-5-Acetylamino-5-(2-methylamino-ethylcarbamoyl)-pentyl]-carbamicacid tert-butyl-hydromorphone-di-ester (L)

Hydromorphone HCl salt (1.24 g, 3.86 mmol) and DIEA (0.67 mL, 3.86 mmol)were suspended in CHCl₃ (12 mL) and sonicated for 1 h at roomtemperature. 4-Nitro phenylchloroformate (600 mg, 2.97 mmol) was addedto the reaction mixture and was then sonicated for 100 min To theactivated hydromorphone reaction mixture was added a solution ofcompound J (1.02 g, 2.97 mmol) and HOBt (0.52 g, 3.86 mmol) in DMF (12mL) dropwise and stirred at room temperature overnight (˜18 h). Solventswere then removed in vacuo and the residue was dissolved in a minimumamount of MeOH and precipitated with an excess of Et₂O. The precipitatewas filtered off, washed with Et₂O and dried under vacuo to affordcompound L. LC-MS [M+H] 656.9 (C₃₄H₄₉N₅O₈+H, calc: 656.7). This crudeproduct was purified by preparative reverse phase HPLC. [Column: VARIAN,LOAD & LOCK, L&L 4002-2 packing: Microsob 100-10 C18, Injection Volume:˜15 mL, Injection flow rate: 20 mL/min, 100% A, (water/0.1% TFA), Flowrate: 100 mL/min, Fraction: 30 Sec (50 mL) Method: 0% B (MeCN/0.1%TFA)/2 min/75% B/96 min/100 ml/min/254 nm]. Pure fractions werecombined, solvents were removed in vacuo. Residue was dried viaco-evaporation with i-PrOH (4×100 mL) to afford compound L as yellow oil(0.90 g, 46%).

Synthesis of (S)-2-Acetylamino-6-amino-hexanoic acid(2-methylamino-ethyl)-amide hydromorphone ester (Compound 3)

Compound L (0.90 g, 1.37 mmol) was suspended in dioxane (˜2 mL),sonicated and treated with 4.0 N HO/dioxane (˜20 mL) at roomtemperature. White precipitate was formed immediately. Next the mixturewas diluted with Et₂O (200 mL), hexane (20 mL) and the precipitate wasfiltered off and washed with Et₂O (100 mL), hexane (100 mL) and driedunder vacuum to afford Compound 3 (0.67 g, 78% yield, 97.5% purity).LC-MS [M+H] 556.3 (C₂₉H₄₁N₅O₆+H, calc: 556.6).

Example 13 Synthesis of[2-((S)-2-Acetylamino-5-guanidino-pentanoylamino)-ethyl]-ethyl-carbamicacid hydromorphone ester (Compound 4)

Preparation 12 Synthesis of2,2,2-trifluoro-N-(2-ethylamino-ethyl)-acetamide (M)

A solution of N-ethylethylenediamine (10.0 g, 113.4 mmol) and ethyltrifluoroacetate (32.0 ml, 261 mmol) in the mixture of acetonitrile (110ml) and water (2.5 ml, 139 mmol) was refluxed with stirring overnight(˜18 h). Solvents were evaporated in vacuo. Residue was re-evaporatedwith i-PrOH (3×100 ml). Residue was dissolved in dichloromethane (500ml) and left overnight at room temperature. The formed crystals werefiltered, washed with dichloromethane (100 ml) and dried in vacuo toprovide compound M (24.6 g, 82.4 mmol) as white solid powder.

Preparation 13 Synthesis of{ethyl-[2-(2,2,2-trifluoro-acetylamino)-ethyl]carbamic acid benzyl ester(N)

A solution of compound M (24.6 g, 82.4 mmol) and DIEA (14.3 ml, 82.4mmol) in THF (100 ml) was cooled to ˜5° C., followed by the addition ofa solution of N-(benzyloxycarbonyl)succinimide (20.3 g, 81.6 mmol) inTHF (75 ml) dropwise over 20 min. The temperature of the reactionmixture was raised to room temperature and stirring was continued for anadditional 30 min. Solvents were evaporated and the residue wasdissolved in EtOAc (500 ml). The organic layer was extracted with 5%aqueous NaHCO₃ (2×100 ml) and brine (100 ml). The organic layer wasevaporated to provide compound N (24.9 g, 78.2 mmol) as yellowish oil.LC-MS [M+H] 319.0 (C₁₄H₁₇F₃N₂O₃+H, calc: 319.2). Compound N was usedwithout further purification.

Preparation 14 Synthesis of (2-Amino-ethyl)-ethyl-carbamic acid benzylester (O)

To a solution of compound N (24.9 g, 78.2 mmol) in MeOH (300 ml) wasadded a solution of LiOH (3.8 g, 156 mmol) in water (30 ml). Thereaction mixture was stirred at room temperature for 5 h. Next thesolvents were evaporated to ¾ of initial volume followed by the dilutionwith water (200 ml). The solution was extracted with EtOAc (200 ml×2)and the organic layer was washed with brine (100 ml), dried over MgSO₄and evaporated in vacuo. Residue was dissolved in ether (200 ml) andtreated with 2 N HCl/ether (200 ml). The formed precipitate wasfiltered, washed with ether and dried in vacuum to provide hydrochloridesalt of compound O (12.1 g, 46.7 mmol) as white solid. LC-MS [M+H] 222.9(C₁₂H₁₈N₂O₂+H, calc: 223.2).

Preparation 15 Synthesis of {2-[boc-Arg(Pbf)]-aminoethyl}-ethyl-carbamicacid benzyl ester (P)

A solution of Boc-Arg(Pbf)-OH (3.0 g, 5.69 mmol), compound O (1.62 g,6.26 mmol), DIEA (3.17 ml, 18.21 mmol) and HATU (2.59 g, 6.83 mmol) inDMF (20 ml) was stirred at room temperature for 1 h. The reactionmixture was diluted with EtOAc (300 ml) and extracted with water (3×75ml) and brine (75 ml). The organic layer was dried over MgSO₄, filteredand then evaporated to provide compound P (5.97 g, yield exceededquantitative) as yellowish oil. LC-MS [M+H] 731.5 (C₃₆H₅₄N₆O₈S+H, calc:731.7). Compound P was used without further purification.

Preparation 16 Synthesis of {2-[H-Arg(Pbf)]-aminoethyl}-ethyl-carbamicacid benzyl ester (Q)

Compound P (5.69 mmol) was dissolved in dioxane (20 ml) and treated with4 N HCl/dioxane (100 ml, 70 mmol) at room temperature for 1 h. Thesolvent was then removed in vacuo, followed by suspension in i-PrOH (50ml) and finally, the solvent was evaporated to remove residual solvents(procedure was repeated twice). The crude reaction mixture was dried invacuo to provide compound Q (5.97, yield exceeded quantitative) asyellowish solid. LC-MS [M+H] 631.5 (C₃₁H₄₆N₆O₆S+H, calc: 631.2).Compound Q was used without further purification.

Preparation 17 Synthesis of {2-[Ac-Arg(Pbf)]-aminoethyl}-ethyl-carbamicacid benzyl ester (R)

A solution of compound Q (5.69 mmol), Ac₂O (649 μl, 6.83 mmol) and DIEA(2.97 ml, 17.07 mmol) in chloroform (20 ml) was stirred at roomtemperature for 1 h. This was followed by addition of 2M EtNH₂/THF (1.71ml, 3.41 mmol). The reaction mixture was stirred at room temperature foran additional 30 min, followed by the dilution with EtOAc (300 ml). Theorganic layer was extracted with water (75 ml), 2% aq. H₂SO₄ (75 ml),water (3×75 ml) and brine (75 ml). The organic layer was then dried overMgSO₄ and evaporated to provide compound R (3.99 g, yield exceededquantitative) as yellowish solid. LC-MS [M+H] 673.6 (C₃₃H₄₈N₆O₇S+H,calc: 672.9). Compound R was used without further purification.

Preparation 18 Synthesis of N-[Ac-Arg(Pbf)]-N′-ethyl-ethane-1,2-diamine(S)

Compound R (5.69 mmol) was dissolved in methanol (50 ml) followed byaddition of Pd/C (5% wt, 1 g) suspension in water (5 ml). Reactionmixture was subjected to hydrogenation (Parr apparatus, 80 psi) at roomtemperature for 1 h. Upon completion, the catalyst was filtered over padof Celite on sintered glass funnel and washed with methanol. Thefiltrate was evaporated in vacuo to provide the compound S (3.06 g,quantitative yield) as colorless oil. LC-MS [M+H] 539.5 (C₂₅H₄₂N₆O₅S+H,calc: 539.9). Compound S was used without further purification.

Synthesis of[2-(2-Acetylamino-5-guanidino-pentanoylamino)-ethyl]-ethyl-carbamic acidhydromorphone ester (Compound 4)

A suspension of hydromorphone hydrochloride (2.75 g, 8.54 mmol) and DIEA(1.49 ml, 8.54 mmol) in chloroform (8 ml) was sonicated in an ultrasonicbath at room temperature for 1 h, followed by addition of 4-nitrophenylchloroformate (1.38 g, 6.83 mmol). The reaction mixture was sonicated inan ultrasonic bath at room temperature for additional 1 h, followed bythe addition of solution of compound S (3.06 g, 5.69 mmol) and1-hydroxybenzotriazole (1.31 g, 9.67 mmol) in DMF (8 ml). The reactionmixture was stirred overnight (˜18 h) at room temperature, followed bysolvents being evaporated in vacuo. The crude reaction mixture wasdissolved in MeOH (10 ml) and precipitated with ether (500 ml). Theformed precipitate was filtered and dried in vacuo to provide Pbfprotected compound 4 (6.96 g yield exceeded quantitative) as off-whitesolid. LC-MS [M+H] 850.6 (C₄₃H₅₉N₇O₉S+H, requires 850.2).

Pbf protected compound 4 was dissolved in a mixture of 5% m-cresol/TFA(100 ml). The reaction mixture was maintained at room temperature for 1h, followed by dilution with ether (2 L). A precipitate was formed andsubsequently filtered over sintered glass funnel, washed with ether (200ml) and dried in vacuo to provide crude compound 4 (5.2 g, 97%) asoff-white solid. Crude compound 4 (5.2 g, 5.54 mmol) was dissolved inwater (50 ml) and subjected to HPLC purification. Nanosyn-Pack Microsorb(100-10)C-18 column (50×300 mm); flow rate=100 ml/min; injection volume50 ml; mobile phase A: 100% water, 0.1% TFA; mobile phase B: 100%acetonitrile, 0.1% TFA; isocratic elution at 0% B in 5 min., gradientelution to 6% B in 6 min, isocratic elution at 6% B in 13 min, gradientelution from 6% B to 55% B in 76 min; detection at 254 nm]. Fractionscontaining the desired compound were combined and concentrated in vacuo.The residue was dissolved in i-PrOH (50 ml) and evaporated in vacuo(procedure was repeated twice). The residue was dissolved in i-PrOH (50ml) and treated with 2 N HCl/ether (200 ml, 400 mmol) to providehydrochloride salt of Compound 4 (1.26 g, 32% yield, 95.7% purity) aswhite solid. LC-MS [M+H] 598.4 (C₃₀H₄₃N₇O₆+H, calc: 598.7). Retentiontime*: 2.53 min

*-[Chromolith SpeedRod RP-18e C18 column (4.6×50 mm); flow rate 1.5ml/min; mobile phase A: 0.1% TFA/water; mobile phase B 0.1% TFA/ACN;gradient elution from 5% B to 100% B over 9.6 min, detection 254 nm]

Example 14 Synthesis of (S)-ethyl4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 101)

Preparation 19 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-pentanoyl]-piperazine-1-carboxylicacid tert-butyl ester (T)

To a solution of Fmoc-Arg(Pbf)-OH 1 (25.0 g, 38.5 mmol) in DMF (200 mL)at room temperature was added DIEA (13.41 mL, 77.1 mmol). After stirringat room temperature for 10 min, the reaction mixture was cooled to ˜5°C. To the reaction mixture was added HATU (16.11 g, 42.4 mmol) inportions and stirred for 20 min and a solution oftert-butyl-1-piperazine carboxylate (7.18 g, 38.5 mmol) in DMF (50 mL)was added dropwise. The reaction mixture was stirred at ˜5° C. for 5min. The mixture reaction was then allowed to warm to room temperatureand stirred for 2 h. Solvent was removed in vacuo and the residue wasdissolved in EtOAc (500 mL), washed with water (2×750 mL), 1% H₂SO₄ (300mL) and brine (750 mL). The organic layer was separated, dried overNa₂SO₄ and solvent removed in vacuo to a total volume of 100 mL.Compound T was taken to the next step as EtOAc solution (100 mL). LC-MS[M+H] 817.5 (C₄₃H₅₆N₆O₈S+H, calc: 817.4).

Preparation 20 Synthesis of4-[(S)-2-Amino-5-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-pentanoyl]-piperazine-1-carboxylicacid tert-butyl ester (U)

To a solution of compound T (46.2 mmol) in EtOAc (175 mL) at roomtemperature was added piperidine (4.57 mL, 46.2 mmol) and the reactionmixture was stirred for 18 h at room temperature. Next the solvent wasremoved in vacuo and the resulting residue dissolved in minimum amountof EtOAc (˜50 mL) and hexane (˜1 L) was added. Precipitated crudeproduct was filtered off and recrystallised again with EtOAc (˜30 mL)and hexane (˜750 mL). The precipitate was filtered off, washed withhexane and dried in vacuo to afford compound U (28.0 g, 46.2 mmol).LC-MS [M+H] 595.4 (C₂₈H₄₆N₆O₆S+H, calc: 595.3).

Preparation 21 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]piperazine-1-carboxylicacid tert-butyl ester (V)

To a solution of compound U (28.0 g, 46.2 mmol) in THF (250 mL) wasadded aqueous 1N NaOH (171 mL). The reaction mixture was cooled to ˜5°C., a solution of 2-naphthalene sulfonylchloride (26.19 g, 115.6 mmol)in THF (125 mL) was added dropwise. The reaction mixture was stirred at˜5° C. for 10 min, with stirring continued at room temperature for 2 h.The reaction mixture was diluted with EtOAc (1 L), washed with aqueous1N NaOH (1 L), water (1 L) and brine (1 L). The organic layer wasseparated, dried over Na₂SO₄ and removal of the solvent in vacuo toafford compound V (36.6 g, 46.2 mmol). LC-MS [M+H] 785.5(C₃₈H₅₂N₆O₈S₂+H, calc: 785.9).

Preparation 22 Synthesis of2,2,4,6,7-Pentamethyl-2,3-dihydro-benzofuran-5-sulfonic acid1-amino-1-[(S)-4-(naphthalene-2-sulfonylamino)-5-oxo-5-piperazin-1-yl-pentylamino]-meth-(E)-ylideneamide(W)

To a solution of compound V (36.6 g, 46.2 mmol) in dioxane (60 mL) wasadded 4M HCl in dioxane (58 mL) dropwise. The reaction mixture wasstirred at room temperature for 1.5 h. Et₂O (600 mL) was added to thereaction mixture, precipitated product was filtered off, washed withEt₂O and finally dried under vacuum to afford compound W (34.5 g, 46.2mmol). LC-MS [M+H] 685.4 (C₃₃H₄₄N₆O₆S₂+H, calc: 685.9). Compound W wasused without further purification.

Preparation 23 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazine-1-carboxylicacid ethyl ester (X)

To a solution of compound W (8.0 g, 11.1 mmol) in CHCl₃ (50 ml) wasadded DIEA (4.1 mL, 23.3 mmol) at room temperature and stirred for 15min. The mixture was cooled to ˜5° C., ethyl chloroformate (1.06 mL,11.1 mmol) was added drop wise. After stirring at room temperatureovernight (˜18 h), solvent removed in vacuo. The residue was dissolvedin MeOH (˜25 ml) and Et2O (˜500 mL) was added. The precipitated crudeproduct was filtered off, washed with Et₂O and dried under vacuo toafford compound X (8.5 g, 11.1 mmol). LC-MS [M+H] 757.6 (C₃₆H₄₈N₆O₈S₂+H,calc: 757.9). Compound X was used without further purification.

Synthesis of (S)-ethyl4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 101)

A solution of 5% m-cresol/TFA (50 ml) was added to compound X (8.5 g,11.1 mmol) at room temperature. After stirring for 1 h, the reactionmixture was precipitated with Et₂O (˜500 mL). The precipitate wasfiltered and washed with Et₂O and dried under vacuo to afford the crudeproduct. The crude product was purified by preparative reverse phaseHPLC. [Column: VARIAN, LOAD & LOCK, L&L 4002-2, Packing: Microsob 100-10C18, Injection, Volume: ˜15 mL x 2, Injection flow rate: 20 mL/min, 100%A, (water/0.1% TFA), Flow rate: 100 mL/min, Fraction: 30 Sec (50 mL),Method: 0% B (MeCN/0.1% TFA)-60% B/60 min/100 ml/min/254 nm]. Solventswere removed from pure fractions in vacuo. Trace of water was removed byco-evaporation with 2× i-PrOH (50 ml). The residue was dissolved in aminimum amount of i-PrOH and product was precipitated with 2 M HCl inEt₂O. Product was filtered off and washed with Et₂O and dried undervacuo to afford Compound 101 as HCl salt 7 (3.78 g, 63% yield, 99.4%purity). LC-MS [M+H] 505.4 (C₃₈H₅₂N₆O₈S₂+H, calc: 505.6).

Example 15 Synthesis of (S)-ethyl4-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 102)

Preparation 24 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-tert-butoxycarbonylamino-pentanoyl]piperazine-1-carboxylicacid ethyl ester (Y)

To a solution of Boc-Arg(Pbf)-OH (13.3 g, 25.3 mmol) in DMF (10 mL) wasadded DIEA (22.0 mL, 126.5 mmol) at room temperature and stirred for 15min. The reaction mixture was then cooled to ˜5° C. and HATU (11.5 g,30.3 mmol) was added in portions and stirred for 30 min, followed by thedropwise addition of ethyl-1-piperazine carboxylate (4.0 g, 25.3 mmol)in DMF (30 mL). After 40 min, the reaction mixture was diluted withEtOAc (400 mL) and poured in to H₂O (1 L). Extracted with EtOAc (2×400mL) and washed with H₂O (800 mL), 2% H₂SO₄ (500 mL), H₂O (2×800 mL) andbrine (800 mL). Organic layer was separated, dried over MgSO₄ andsolvent removed in vacuo. The resultant oily residue was dried in vacuoto afford compound Y (16.4 g, 24.5 mmol) as foamy solid. LC-MS [M+H]667.2 (C₃₁H₅₀N₆O₈S+H, calc: 667.8). Compound Y was used without furtherpurification.

Preparation 25 Synthesis of4-[(S)-2-Amino-5-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5sulfonylimino]-methyl}amino)-pentanoyl]-piperazine-1-carboxylic acidethyl ester (Z)

A solution of compound Y (20.2 g, 30.2 mmol) in dichloromethane (90 mL)was treated with 4.0 N HCl in 1,4-dioxane (90 mL, 363.3 mmol) andstirred at room temperature for 2 h. Next most of the dichloromethanewas removed in vacuo and Et₂O (˜1 L) was added. The resultantprecipitate was filtered off and washed with Et₂O and dried in vacuo toafford compound Z (17.8 g, 30.2 mmol). LC-MS [1\4+1-1] 567.8(C₂₆H₄₂N₆O₆S+H, calc: 567.8).

Preparation 26 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}amino)-2-(2,4,6-triisopropyl-benzenesulfonylamino)-pentanoyl]-piperazine-1-carboxylicacid ethyl ester (AA)

To a solution of compound Z (1.0 g, 1.8 mmol) in THF (7 mL) was added3.1N aqueous NaOH (4.0 mL) and stirred for 5 min. The reaction mixturewas cooled to ˜5° C., and then a solution of tripsyl chloride added dropwise (2.2 g, 7.3 mmol) in THF (5 mL) and stirred at room temperatureovernight (˜18 h). The reaction mixture was diluted with H₂O (130 mL),acidified with 2% H₂SO₄ (15 mL) and extracted with EtOAc (3×80 mL).Organic layer were combined and washed with H₂O (2×400 mL), saturatedNaHCO₃ (100 mL), H₂O (200 mL) and brine (200 mL). The organic layer wasseparated dried over MgSO₄ and solvent removed in vacuo to afford (2.9g) of crude product. This was purified by normal phase flashchromatography (5-10% MeOH/DCM) to afford compound AA (0.52 g, 1.0mmol). LC-MS [M+H] 833.8 (C₄₁H₆₄N₆O₈S₂+H, calc: 834.1).

Synthesis of (S)-ethyl4-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 102)

A solution of 5% m-cresol/TFA (40 ml) was added to compound AA (3.73 g,3.32 mmol) at room temperature. After stirring for 45 min, solvents wereremoved in vacuo. Residue was dissolved in dichloromethane (100 ml),washed with H₂O (3×200 mL) and brine (200 mL). The organic layer wasseparated, dried over MgSO₄ and then the solvent removed in vacuo. Theresidue was dissolved in dichloromethane (˜5 mL) and then hexane (˜250mL) was added and a precipitate was formed. This was washed with hexaneand dried under vacuo to afford the crude product (1.95 g). The crudeproduct was purified by reverse phase HPLC [Column: VARIAN, LOAD & LOCK,L&L 4002-2, Packing: Microsob 100-10 C18, Injection Volume: ˜15 mL,Injection flow rate: 20 mL/min, 100% A, (water/0.1% TFA), Flow rate: 100mL/min, Fraction: 30 Sec (50 mL), Method: 25% B (MeCN/0.1% TFA)/70% B/98min/100 ml/min/254 nm]. Solvents were removed from pure fractions invacuo. Trace of water was removed by co-evaporation with 2×i-PrOH (50ml). The residue was dissolved in a minimum amount of i-PrOH and productwas precipitated with 2 M HCl in Et₂O. Product was filtered off andwashed with Et₂O and dried under vacuo to afford the product as HCl saltof Compound 102 (0.72 g, 35% yield, 99.8% purity). LC-MS [M+1-1] 581.6(C₂₈H₄₈N₆O₅S+H, calc: 581.7).

Example 16 Synthesis of (S)-ethyl1-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 103)

Preparation 27 Synthesis of 1-[boc-Arg(Pbf)]-piperidine-4-carboxylicacid ethyl ester (BB)

To a solution of Boc-Arg(Pbf)-OH (3.4 g, 6.36 mmol) and HATU (2.9 g,7.63 mmol) in DMF (15 mL) was added DIEA (7.4 mL, 42.4 mmol) and thereaction mixture was stirred for 10 min at room temperature. A solutionof ethyl isonipecotate (1.0 g, 6.36 mmol) in DMF (6 mL) was added to thereaction mixture dropwise. The reaction mixture was stirred at roomtemperature for 1 h, then diluted with ethyl acetate (150 mL) and pouredinto water (500 mL). The product was extracted with ethyl acetate (2×100mL). Organic layer was washed with aqueous 0.1 N HCl (200 mL), 2%aqueous sodium bicarbonate (200 mL), water (200 mL) and brine (200 mL).The organic layer was dried over sodium sulfate, filtered, and thenevaporated in vacuo. The resultant oily product was dried in vacuoovernight to give compound BB (3.7 g, 5.57 mmol) as a viscous solid.LC-MS [M+H] 666.5 (C₃₂H₅₁N₅O₈S+H, calc: 666.7). Compound BB was usedwithout further purification.

Preparation 28 Synthesis of 1-[Arg(Pbf)]-piperidine-4-carboxylic acidethyl ester HCl salt (CC)

To a solution of compound BB (4.7 g, 7.07 mmol) in dichloromethane (25mL) was added 4N HCl in dioxane (25.0 mL, 84.84 mmol), and the reactionmixture was stirred at room temperature for 2 h. The reaction mixturewas concentrated in vacuo to ˜20 mL of solvent, and then diluted withdiethyl ether (250 mL) to produce a white fine precipitate. The reactionmixture was stirred for 1 h and the solid was washed with ether (50 mL)and dried in a high vacuum overnight to give compound CC (4.3 g, 7.07mmol) as a fine powder. LC-MS [M+H] 566.5 (C₂₇H₄₃N₅O₆S+H, calc: 566.7).

Preparation 29 Synthesis of1-[5(S)—(N′-Pbf-guanidino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperidine-4-carboxylicacid ethyl ester (DD)

To a solution of compound CC (1.1 g, 1.6 mmol) and NaOH (260 mg, 5.9mmol) in a mixture of THF (5 mL) and water (3 mL) was added a solutionof 2-naphthalosulfonyl chloride (0.91 g, 2.5 mmol) in THF (10 mL)dropwise with stirring at ˜5° C. The reaction mixture was stirred atroom temperature for 1 h, then diluted with water (5 mL). Aqueous 1N HCl(5 mL) was added to obtain pH ˜3. Additional water was added (20 mL),and the product was extracted with ethyl acetate (3×50 mL). The organiclayer was removed and then washed with 2% aqueous sodium bicarbonate (50mL), water (50 mL) and brine (50 mL). The extract was dried overanhydrous sodium sulfate, filtered, and was evaporated in vacuo. Theformed oily product was dried in vacuo overnight to give compound DD(1.3 g, 1.6 mmol) as an oily foaming solid. LC-MS [M+H] 756.5(C₃₇H₄₉N₅O₈S₂+H, calc: 756.7).

Synthesis of (S)-ethyl1-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 103)

To a flask, was added compound DD (1.3 g, 1.6 mmol) and then treatedwith 5% m-cresol/TFA (10 mL). The reaction mixture was stirred at roomtemperature for 1 h. Next, the reaction mixture was concentrated invacuo to a volume 5 mL. Diethyl ether (200 mL) was then added to theresidue, and formed fine white precipitate. The precipitate was filteredoff and washed with ether (2×25 mL). The resultant solid was dried invacuo overnight to give a crude material, which was purified bypreparative reverse phase HPLC. [Nanosyn-Pack Microsorb (100-10)C-18column (50×300 mm); flow rate=100 ml/min; injection volume 12 ml(DMSO-water, 1:1, v/v); mobile phase A: 100% water, 0.1% TFA; mobilephase B: 100% ACN, 0.1% TFA; gradient elution from 25% B to 55% B in 90min, detection at 254 nm]. Fractions containing desired compound werecombined and concentrated in vacuo. The residue was dissolved in i-PrOH(50 ml) and evaporated in vacuum (repeated twice). The residue was nextdissolved in i-PrOH (5 ml) and treated with 2 N HCl/ether (100 ml, 200mmol) to give a white precipitate. It was dried in vacuo overnight togive Compound 103 (306 mg, 31% yield, 95.7% purity) as a white solid.LC-MS [M+H] 504.5 (C₂₄H₃₃N₅O₅S+H, calc: 504.6).

Example 17 Synthesis of (S)-ethyl1-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 104)

Preparation 30 Synthesis of1-[5(S)—(N′-Pbf-guanidino)-2-(2,4,6-triisopropyl-benzenesulfonylamino)-pentanoyl]-piperidine-4-carboxylicacid ethyl ester (EE)

To a solution of compound CC (1.0 g, 1.6 mmol) and NaOH (420.0 mg, 10.4mmol) in a mixture of THF (5 mL) and water (4 mL) was added a solutionof 2,4,6-triisopropyl-benzenesulfonyl chloride (2.4 g, 8.0 mmol) dropwise with stirring and maintained at ˜5° C. The reaction mixture wasthen stirred at room temperature for 1 h, monitoring the reactionprogress, then diluted with water (20 mL), and acidified with aqueous 1N HCl (5 mL) to pH ˜3. Additional water was added (30 mL), and theproduct was extracted with ethyl acetate (3×50 mL). The organic layerwas washed with 2% aqueous sodium bicarbonate (50 mL), water (50 mL) andbrine (50 mL). The organic layer was dried over anhydrous sodiumsulfate, filtered, and was evaporated in vacuo. Formed oily residue wasdried in a vacuo overnight to give compound EE (1.0 g, 1.2 mmol) as anoily material. LC-MS [M+H] 832.8 (C₄₂H₆₅N₅O₈S₂+H, calc: 832.7).

Synthesis of (S)-ethyl1-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 104)

To a flask was added compound EE (2.3 g, 2.8 mmol) and then treated with5% m-cresol/TFA (16 mL). The reaction mixture was stirred at roomtemperature for 1 h. The reaction mixture was then concentrated in vacuoto a volume of 5 mL. Hexane (200 mL) was added to the residue anddecanted off to give an oily precipitate. The product was purified bypreparative reverse phase HPLC. Nanosyn-Pack Microsorb (100-10)C-18column (50×300 mm); flow rate=100 ml/min; injection volume 15 ml(DMSO-water, 1:1, v/v); mobile phase A: 100% water, 0.1% TFA; mobilephase B: 100% ACN, 0.1% TFA; gradient elution from 35% B to 70% B in 90min, detection at 254 nm]. Fractions containing desired compound werecombined and concentrated in vacuo. The residue was dissolved in i-PrOH(100 ml) and evaporated in vacuo (repeated twice). The residue wasdissolved in i-PrOH (5 ml) and treated with 2 N HCl/ether (100 ml, 200mmol) to give an oily residue. It was dried in vacuo overnight to giveCompound 104 (1.08 g, 62.8%) as a viscous solid. LC-MS [M+1-1] 580.6(C₂₉H₄₉N₅O₅S+H, calc: 580.8).

Example 18 Synthesis of(S)-6-(4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoicacid (Compound 105)

Preparation 31 Synthesis of6-{4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazin-1-yl}-6-oxo-hexanoicacid methyl ester (FF)

To a solution of compound W (1.5 g, 2.08 mmol) in CHCl₃ (50 mL) wasadded DIEA (1.21 mL, 4.16 mmol) followed by adipoyl chloride (0.83 mL,6.93 mmol) dropwise. The reaction mixture was stirred at roomtemperature overnight (˜18 h). Solvents were removed in vacuo and theresidue was dried under vacuo to afford the compound FF (2.1 g, amountexceeds quantative). LC-MS [M+H] 827.5 (C₄₀H₅₄N₆O₉S₂+H, calc: 827.3).Compound FF was used without further purification.

Preparation 32 Synthesis of6-{4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazin-1-yl}-6-oxohexanoicacid (GG)

To a solution of compound FF (2.1 g, 2.08 mmol) in THF (5 mL), H₂O (5mL) was added 2 M aq LiOH (6 mL). The reaction mixture was stirred atroom temperature for 2 h. Solvents were removed in vacuo, then theresidue was dissolved in water (˜50 mL), acidified with saturatedaqueous NaHSO₄ (˜100 ml) and extracted with EtOAc (2×100 ml). Theorganic layer was dried over Na₂SO₄ and removal of the solvent gavecompound GG (1.72 g, 2.08 mmol). LC-MS [M+H] 813.5 (C₃₉H₅₂N₆O₉S₂+H,calc: 813.3). Compound GG was used without further purification.

Synthesis of(S)-6-(4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoicacid (Compound 105)

A solution of 5% m-cresol/TFA (25 ml) was added to compound GG (1.72 g,2.08 mmol) at room temperature. After stirring for 30 min, the reactionmixture was precipitated with addition of Et₂O (˜200 mL). Theprecipitate was filtered and washed with Et₂O and dried under vacuo toafford the crude product. The crude product was purified by preparativereverse phase HPLC [Column: VARIAN, LOAD & LOCK, L&L 4002-2, Packing:Microsob 100-10 C18, Injection Volume: ˜25 mL, Injection flow rate: 20mL/min, 95% A, (water/0.1% TFA), Flow rate: 100 mL/min, Fraction: 30 Sec(50 mL), Method: 5% B (MeCN/0.1% TFA)/5 min/25% B/20 min/25% B/15min/50% B/25 min/100 ml/min/254 nm]. Solvents were removed from purefractions in vacuo. Trace of water was removed by co-evaporation withi-PrOH (25 ml) (repeated twice). The residue was dissolved in a minimumamount of i-PrOH, then 2 M HCl in Et₂O (˜50 mL) was added and dilutedwith Et₂O (˜250 mL). Precipitate formed was filtered off and washed withEt₂O and dried under vacuo to afford the product as HCl salt Compound105 (0.74 g, 59% yield, 98.9% purity). LC-MS [M+H] 561.4 (C₂₆H₃₆N₆O₆S+H,calc: 561.2).

Example 19 Synthesis of 3-(4-carbamimidoylphenyl)-2-oxopropanoic acid(Compound 107)

Compound 107, i.e., 3-(4-carbamimidoylphenyl)-2-oxopropanoic acid can beproduced using methods known to those skilled in the art, such as thatdescribed by Richter P et al, Pharmazie, 1977, 32, 216-220 andreferences contained within. The purity of Compound 107 used in Example7 was estimated to be 76%, an estimate due low UV absorbance of thiscompound via HPLC. Mass spec data: LC-MS [M+H] 207.0 (C10H10N2O3+H,calc: 207.1).

Example 20 Synthesis of(S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butanamido)pentanoicacid (Compound 108)

Preparation 33 Synthesis of(S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acidbenzyl ester (HH)

A solution of Boc-Glu(OBz1)-OH (7.08 g, 21.0 mmol), BOP (9.72 g, 22.0mmol) and DIEA (12.18 ml, 70.0 mmol) in DMF (50 ml) was maintained atroom temperature for 20 min, followed by the addition of4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). Thereaction mixture was stirred at room temperature for an additional 1 hand diluted with EtOAc (500 ml). The obtained solution was extractedwith water (100 ml), 5% aq. NaHCO₃ (100 ml) and water (2×100 ml). Theorganic layer was dried over MgSO₄, evaporated and dried in vacuo toprovide compound HH (9.65 g, yield exceeded quantitative) as yellowishoil. LC-MS [M+H] 452.0 (C₂₅H₂₉N₃O₅+H, calc: 452.4). Compound HH was usedwithout further purification.

Preparation 34 Synthesis of(S)-4-tert-butoxycarbonylamino-4-[4-(N-hydroxycarbamimidoyl)-benzylcarbamoyl]-butyric acid benzyl ester (II)

A solution of compound HH (9.65 g, 20.0 mmol), hydroxylaminehydrochloride (2.10 g, 30.0 mmol) and DIEA (5.22 ml, 30.0 mmol) inethanol (abs., 150 ml) was refluxed for 6 h. The reaction mixture wasallowed to cool to room temperature and stirred for additional 16 h,then the solvents were evaporated in vacuo. The resultant residue wasdried in vacuo to provide compound II (14.8 g, yield exceededquantitative) as yellowish oil. LC-MS [M+H] 485.5 (C₂₅H₃₂N₄O₆+H, calc:485.8). Compound II was used without further purification.

Preparation 35 Synthesis of(S)-4-tert-butoxycarbonylamino-4-[4-(N-acetylhydroxycarbamimidoyl)-benzylcarbamoyl]-butyric acid benzyl ester (JJ)

A solution of compound II (14.8 g, 20.0 mmol) and acetic anhydride (5.7ml, 60.0 mmol) in acetic acid (100 ml) was stirred at room temperaturefor 45 min, and then solvent was evaporated in vacuo. The resultantresidue was dissolved in EtOAc (300 ml) and extracted with water (2×75ml) and brine (75 ml). The organic layer was then dried over MgSO₄,evaporated and dried in vacuo to provide compound JJ (9.58 g, 18.2 mmol)as yellowish solid. LC-MS [M+H] 527.6 (C₂₇H₃₄N₄O₇+H, calc: 527.9).Compound JJ was used without further purification.

Preparation 36 Synthesis of(S)-4-[4-(N-acetylhydroxycarbamimidoyl)-benzyl carbamoyl]-butyric acidbenzyl ester (KK)

Compound JJ (9.58 g, 18.2 mmol) was dissolved in 1,4-dioxane (50 ml) andtreated with 4 N HCl/dioxane (50 ml, 200 mmol) at room temperature for 1h. Next, the solvent was evaporated in vacuo. The resultant residue wastriturated with ether (200 ml). The obtained precipitate was filtrated,washed with ether (100 ml) and hexane (50 ml) and dried in vacuo toprovide compound KK (9.64 g, yield exceeded quantitative) as off-whitesolid. LC-MS [M+H] 426.9 (C₂₂H₂₆N₄O₅+H, calc: 427.3). Compound KK wasused without further purification.

Preparation 37 Synthesis of(R)-4-phenyl-2-phenylmethanesulfonylamino-butyric acid (LL)

A solution of D-homo-phenylalanine (10.0 g, 55.9 mmol) and NaOH (3.35 g,83.8 mmol) in a mixture of 1,4-dioxane (80 ml) and water (50 ml) wascooled to −5° C., followed by alternate addition of α-toluenesulfonylchloride (16.0 g, 83.8 mmol; 5 portions by 3.2 g) and 1.12 M NaOH (50ml, 55.9 mmol; 5 portions by 10 ml) maintaining pH >10. The reactionmixture was acidified with 2% aq. H₂SO₄ to pH=−2. The obtained solutionwas extracted with EtOAc (2×200 ml). The organic layer was washed withwater (3×75 ml), dried over MgSO₄ and then the solvent was evaporated invacuo. The resultant residue was dried in vacuo to provide compound LL(12.6 g, 37.5 mmol) as white solid. LC-MS [M+H] 334.2 (C₁₇H₁₉NO₄S+H,calc: 333.4). Compound LL was used without further purification.

Preparation 38 Synthesis of(S)-4-[4-(N-acetylhydroxycarbamimidoyl)-benzylcarbamoyl]-4-((R)-4-phenyl-2-phenylmethanesulfonylamino-butyrylamino)-butyricacid benzyl ester (MM)

A solution of compound LL (5.9 g, 17.8 mmol), compound KKdi-hydrochloride (18.0 mmol), BOP (8.65 g, 19.6 mmol) and DIEA (10.96ml, 19.6 mmol) in DMF (250 ml) was stirred at room temperature for 2 h.The reaction mixture was diluted with EtOAc (750 ml) and extracted withwater (200 ml). The formed precipitate was filtrated, washed with EtOAc(200 ml) and water (200 ml) and dried at room temperature overnight (˜18h) to provide compound MM (8.2 g, 11.0 mmol) as off-white solid. LC-MS[M+H] 743.6 (C₃₉H₄₃N₅O₈S+H, calc: 743.9). Compound MM was used withoutfurther purification.

Synthesis of(S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butanamido)pentanoicacid (Compound 108)

Compound MM (8.0 g, 10.77 mmol) was dissolved in acetic acid (700 ml)followed by the addition of Pd/C (5% wt, 3.0 g) as a suspension in water(50 ml). Reaction mixture was subjected to hydrogenation (Parrapparatus, 5 psi) at room temperature for 3 h. The catalyst was filteredover a pad of Celite on sintered glass filter and washed with methanol.Filtrate was evaporated in vacuo to provide compound 108 as colorlessoil. LC-MS [M+H] 594.2 (C₃₀H₃₅N₅O₆S+H, calc: 594). Obtained oil wasdissolved in water (150 ml) and subjected to HPLC purification.[Nanosyn-Pack YMC-ODS-A (100-10)C-18 column to (75×300 mm); flowrate=250 ml/min; injection volume 150 ml; mobile phase A: 100% water,0.1% TFA; mobile phase B: 100% acetonitrile, 0.1% TFA; isocratic elutionat 10% B in 4 min, gradient elution to 24% B in 18 min, isocraticelution at 24% B in 20 min, gradient elution from 24% B to 58% B in 68min; detection at 254 nm]. Fractions containing desired compound werecombined and concentrated in vacuo. Residue was dissolved in i-PrOH (75ml) and evaporated in vacuo (procedure was repeated twice) to provideCompound 108 (4.5 g, 70% yield, 98.0% purity) as white solid. LC-MS[M+H] 594.2 (C₃₀H₃₅N₅O₆S+H, calc: 594). Retention time*: 3.55 min

*-[Chromolith SpeedRod RP-18e C18 column (4.6×50 mm); flow rate 1.5ml/min; mobile phase A: 0.1% TFA/water; mobile phase B 0.1%TFA/acetonitrile; gradient elution from 5% B to 100% B over 9.6 min,detection 254 nm]

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-48. (canceled)
 49. A pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general formula (II):X—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵)  (II) or apharmaceutically acceptable salt thereof, in which: X represents aresidue of a phenolic opioid selected from the group consisting ofbuprenorphine, dihydroetorphine, diprenorphine, etorphine,hydromorphone, levorphanol, morphine, nalmefene, naloxone,N-methylnaloxone, naltrexone, N-methylnaltrexone, oxymorphone,oripavine, ketobemidone, dezocine, pentazocine, phenazocine,butorphanol, nalbuphine, meptazinol, O-desmethyltramadol, tapentadol,and nalorphine, wherein the hydrogen atom of the phenolic hydroxyl groupis replaced by a covalent bond to—C(O)—NR¹—(C(R²)(R³))_(n)—NH—C(O)—CH(R⁴)—NH(R⁵); R¹ is selected fromalkyl, substituted alkyl, arylalkyl, substituted arylalkyl, aryl andsubstituted aryl; each R² is independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl;each R³ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl; or R² and R³together with the carbon to which they are attached form a cycloalkyl orsubstituted cycloalkyl group, or two R² or R³ groups on adjacent carbonatoms, together with the carbon atoms to which they are attached, form acycloalkyl or substituted cycloalkyl group; n represents an integer from2 to 4; R⁴ represents —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, theconfiguration of the carbon atom to which R⁴ is attached correspondingwith that in an L-amino acid; and R⁵ represents a hydrogen atom, anN-acyl group, a residue of an amino acid, a dipeptide, or an N-acylderivative of an amino acid or dipeptide.
 50. The pharmaceuticalcomposition of claim 49, wherein R¹ represents a (1-4C)alkyl group; 51.The pharmaceutical composition of claim 49, wherein R¹ represents amethyl or ethyl group.
 52. The pharmaceutical composition of claim 49,wherein R¹ represents a methyl group.
 53. The pharmaceutical compositionof claim 49, wherein each of R² and R³ each independently represents ahydrogen atom or a (1-4C)alkyl group;
 54. The pharmaceutical compositionof claim 49, wherein each of R² and R³ represents a hydrogen atom. 55.The pharmaceutical composition of claim 49, wherein R⁵ is acetyl,benzoyl, malonyl, piperonyl, succinyL N-acetylarginine orN-acetyllysine.
 56. The pharmaceutical composition of claim 49, whereinR² and R³ are hydrogen.
 57. The pharmaceutical composition of claim 49,wherein R² and R³ which are on the same carbon are alkyl.
 58. Thepharmaceutical composition of claim 49, wherein R² and R³ which are onthe same carbon form a spirocycle.
 59. The pharmaceutical composition ofclaim 49, wherein R² and R³ which are on the same carbon are methyl. 60.The pharmaceutical composition of claim 49, wherein R² and R³ canmodulate a rate of intramolecular cyclization.
 61. The pharmaceuticalcomposition of claim 49, wherein R² and R³ comprise anelectron-withdrawing group or an electron-donating group.
 62. Thepharmaceutical composition of claim 49, wherein —[C(R²)(R³)]_(n)— isselected from —CH(CH₂F)CH(CH₂F)—; —CH(CHF₂)CH(CHF₂)—; —CH(CF₃)CH(CF₃)—;—CH₂CH(CF₃)—; —CH₂CH(CHF₂)—; —CH₂CH(CH₂F)—; —CH₂CH(F)CH₂—;—CH₂C(F₂)CH₂—; —CH₂CH(C(O)NR²⁰R²¹)—; —CH₂CH(C(O)OR²²)—; —CH₂CH(C(O)OH)—;—CH(CH₂F)CH₂CH(CH₂F)—; —CH(CHF₂)CH₂CH(CHF₂)—; —CH(CF₃)CH₂CH(CF₃)—;—CH₂CH₂CH(CF₃)—; —CH₂CH₂CH(CHF₂)—; —CH₂CH₂CH(CH₂F)—;—CH₂CH₂CH(C(O)NR²³R²⁴)—; —CH₂CH₂CH(C(O)OR²⁵)—; and —CH₂CH₂CH(C(O)OH)—,in which R²°, R²¹, R²² and R²³ each independently represents hydrogen or(1-6C)alkyl, and R²⁴ and R²⁵ each independently represents (1-6C)alkyl.63. The pharmaceutical composition of claim 49, wherein one of R² and R³is aminoacyl.
 64. The pharmaceutical composition of claim 49, whereinone of R² and R³ is

wherein each R¹⁰ is independently selected from hydrogen, alkyl,substituted alkyl, and acyl, and R¹¹ is alkyl or substituted alkyl. 65.The pharmaceutical composition of claim 49, wherein n represents 2 or 3;66. The pharmaceutical composition of claim 49, wherein n represents 2.67. The pharmaceutical composition of claim 49, wherein R⁴ represents—CH₂CH₂CH₂NHC(═NH)(NH₂).
 68. The pharmaceutical composition of claim 49,wherein R⁵ represents an N-acyl group.
 69. The pharmaceuticalcomposition of claim 49, wherein acyl is substituted acyl.
 70. Thepharmaceutical composition of claim 68, wherein the N-acyl group is anN-(1-4C)alkanoyl, N-benzoyl or N-piperonyl group.
 71. The pharmaceuticalcomposition of claim 68, wherein R⁵ is acetyl, benzoyl, malonyl,piperonyl, succinyl, N-acetylarginine or N-acetyllysine.
 72. Thepharmaceutical composition of claim 49, wherein R⁵ is an acetyl,glycinyl or N-acetylglycinyl group.
 73. The pharmaceutical compositionof claim 72, wherein R⁵ is an acetyl group.
 74. The pharmaceuticalcomposition of claim 49, wherein the group —C(O)—CH(R⁴)—NH(R⁵) isN-acetylarginine.
 75. The pharmaceutical composition of claim 49,wherein the trypsin inhibitor is derived from soybean.
 76. Thepharmaceutical composition of claim 49, wherein the trypsin inhibitor isan arginine mimic or a lysine mimic.
 77. The pharmaceutical compositionof claim 76, wherein the arginine mimic or lysine mimic is a syntheticcompound.
 78. The pharmaceutical composition of claim 49, wherein thetrypsin inhibitor is a compound of formula: wherein:

Q¹ is selected from —O-Q⁴ or -Q⁴-COOH, where Q⁴ is C₁-C₄ alkyl; Q² is Nor CH; and Q³ is aryl or substituted aryl.
 79. The pharmaceuticalcomposition of claim 49, wherein the trypsin inhibitor is a compound offormula: wherein:

Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where Q⁶ is —(CH₂)_(p)—COOH; Q⁷is —(CH₂)—C₆H₅; and p is an integer from one to three; and r is aninteger from one to three.
 80. The pharmaceutical composition of claim49, wherein the trypsin inhibitor is selected from (S)-ethyl4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazine-1-carboxylate;(S)-ethyl4-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate;(S)-ethyl1-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperidine-4-carboxylate;(S)-ethyl1-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylate;(S)-6-(4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoicacid; 4-aminobenzimidamide; 3-(4-carbamimidoylphenyl)-2-oxopropanoicacid;(S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butanamido)pentanoicacid; 6-carbamimidoylnaphthalen-2-yl 4-(diaminomethyleneamino)benzoate;and 4,4′-(pentane-1,5-diylbis(oxy))dibenzimidamide.
 81. Thepharmaceutical composition of claim 49, wherein the trypsin inhibitor is6-carbamimidoylnaphthalen-2-yl 4-(diaminomethyleneamino)benzoate(Compound 109).